1. Extraction and Sample Purification
Applied Chromatography
Dr. Lina Dahabiyeh

2. Extraction
Chromatography is a separation technique that can be used for qualitative and quantitative analyses.
Most analyses of pharmaceuticals require an extraction step(s).
Extraction methods can be used to separate a substance selectively from a mixture, or to remove unwanted impurities from a solution (ex. Insoluble tablet matrix materials or oily excipients in creams and ointments).
Complex extraction and derivatisation methods are most often applied to bioanalytical measurements and to the concentration of trace impurities of pharmaceuticals rather than to straightforward quality control of active ingredients in pharmaceuticals (active ingredient in a formulation generally utilises a simple extraction procedure).

3. Solvent extraction methods A) Liquid-Liquid Extraction
Liquid-liquid extraction is based on the transfer of a solute substance from one liquid phase into another liquid phase according to the solubility.
Extraction becomes a very useful tool if you choose a suitable extraction solvent.

In the practical use, usually one phase is a water or water-based (aqueous) solution and the other is an organic solvent which is immiscible with water.
The success of this method depends upon the difference in solubility of a compound in various solvents. For a given compound, solubility differences between solvents is quantified as the "distribution coefficient"

5. At a certain temperature, the ratio of concentrations of a solute in each solvent is always constant. This ratio is called the distribution coefficient, K.
(when solvent1 and solvent2 are immiscible liquids)

6. For example, suppose the compound has a distribution coefficient K = 2 between solvent1 (water) and solvent2,(organic solvent)

7. How can we increase the efficiency of extraction
How can we increase the efficiency of extraction??? If you extract twice with 1/2 the volume, the extraction is more efficient than if you extract once with a full volume.
The greater the number of small extractions, the greater the quantity of solute removed.

8. How can organic acids or bases be converted to a water-soluble form?

11. B) Partitioning between organic solvents
Is used in the extraction of analytes from oily excipients such as in the extraction of steroid cream prior to HPLC analysis.
The most commonly used systems are methanol/hexane, aq ethanol/hexane, or acetonitrile/hexane.
Analysis of corticosteroids in creams. The cream is usually dispersed by heating in hexane and then extracted with an equal volume of methanol. Methanol and hexane mix only very slightly and the oily excipients remain predominantly in the hexane layer, while the more polar corticosteroid partitions into the methanol layer.
Ex. from the pharmacopoeial include assays of hydrocortisone acetate cream.

12. Solid-phase extraction (SPE)
SPE is useful for selective separation of interferants from analytes which is not readily achievable by liquid/liquid extraction.
Widely used in bioanalytical measurements and environmental monitoring for concentrating trace amounts of analytes and as a clean up procedure of biological sample prior to analysis.
In principle the analyte is dead stopped on the SPE medium by loading it onto the cartridge in a solvent of low eluting power. It may then be washed with other solvents of low eluting power and is finally eluted with a small volume of a strong solvent.

13. An SPE cartridge
Available in a wide variety of chemistries, absorbents and sizes.
Selecting the most suitable product for each application and sample is important.

14. Methodology

15. A vacuum manifold can be used for conducting multiple extractions simultaneously

16. Advantages in comparison with liquid/liquid extraction
Solid-phase extraction
The solid phase is immiscible with solvents and thus, after loading the sample, a range of washing conditions can be used to remove interferants through having a wide choice of washing solvents.
Chemical nature of adsorbant can be varied so that it is selective for a particular functional group in the analyte.
Emulsions are not formed between the two phases
A sample in a large volume of solution can be trapped on the column (dead stopped) and thus concentrated
Only small volumes of solvent are required for both washing and elution
Extraction can be carried out in batches rather than serially
The expense of the columns can be offset against savings in solvent purchase and disposal.

17. Types of adsorbants used in SPE
Lipophilic silica
Polar surface-modified silica
Ion exchange (anion and cation exchangers based on surface-modified silica).

18. Lipophilic Silica
Involves non-polar modified silica (solid phase, stationary phase)
The lipophilic silica gels will retain lipophilic compounds, provided they are in an un-ionised state, through van der Waals interactions.

19. Basic compounds are adsorbed from aqueous solution by adjusting to alkaline pH with buffer, e.g. a buffer of pH 10 would be suitable for most bases. The column can be washed with further aliquots of alkaline buffer, water. The compound is finally eluted with either an acidic buffer or an organic solvent such as methanol or acetonitrile. Acidic compounds are extracted from aqueous solution by adjusting to acidic pH. The column can be washed with dilute acid, water The compound can be eluted with methanol, acetonitrile, tetrahydrofuron (THF) or alkaline buffer. Neutral compounds can be extracted without controlling pH. Washing can be carried out with dilute acid or alkaline buffer (to remove ionisable impurities) and methanol/water mixtures. The compounds can be eluted from the column with methanol, ethanol or chloroform.

20. Polar surface-modified silica
Retain analytes through interaction between polar groups; silica gel itself or the surface-modified polar silica gels may be used.

21. These adsorbants are typically used for polar compounds that are not well retained by reverse-phase adsorbants.
The columns are conditioned with the solvent which will be used to elute the analyte. The sample is loaded onto the column in a solvent which is not sufficiently strong to elute it.
Washing of the column is often carried out with a moderate-polarity organic solvent, e.g. alcohol-free methylene chloride. Polar compounds are then eluted with methanol or mixtures of methanol and acidic buffer (for basic compounds) or methanol and alkaline buffer (for acidic compounds).
Diol columns have been used to good effect in the extraction of polar drugs from pharmaceutical creams

22. Ion Exchange Chromatography
Retention mechanism is based on electrostatic attraction of the charged functional group on the compound to the charged group that is bonded to the silica surface.
Anionic compounds can be isolated on SAX (strong anion exchanger) or NH2 bonded silica cartridges.
Cationic compounds can be isolated by using SCX (strong cation exchanger) or WCX (weak cation exchanger) bonded silica cartridges

23. Silica-gel-based ion exchange SPE media
The acidic sample is applied in a buffer one or two pH units above its pKa value, e.g. for methicillin. Methicillin can be extracted with either a strong or a weak anion exchanger.
The sample is applied in a buffer one or two pH units below its pKa value, e.g. for the extraction of adrenaline ammonium chloride (NH4Cl) buffer pH 8.3 might be used.

24. pH and Ion Exchange
Retaining a compound by ion exchange from an aqueous solution; pH of sample matrix must be as such at which both the compound of interest and the functional group on the bonded silica are charged.
To elute the compound of interest:
-- A solution with pH which neutralizes either the compounds functional group or the functional group on the sorbents surface is used to elute the compound of interest.
--- Alternatively a solution that has a high ionic strength or that contains an ionic species that displaces the absorbed compound is used.
Weak anions can be extracted and eluted; they can either be displaced by an alternative anion (a buffer containing a counter ion at a high concentration(NaCl) or eluted by ionisation suppression with an acidic solution that neutralises the weak anion.
Also there should be few if any other species of the same charge as the compound in the matrix that may interfere with the adsorption of the compound of interest
Altenatively is used to elute the compound.
When one of these functional groups is neutralised, the electrostatic force that binds the two together is disrupted and the compound is eluted.

25. Factors requiring attention in SPE with silica gels
Too fast flow rate does not allow sufficient time for equilibration between the extraction medium and the solvent flowing through it.
The capacity of sorbent gels is 1–5% of their mass, e.g. for a 100 mg cartridge 1–5 mg.
Non-selective gels may have reduced sample capacity for dirty sample matrices, e.g. if a small amount of octadecyl gel is used to extract a sample from a matrix containing large amounts of lipophilic materials, the gel capacity may be exceeded.
Attention must be paid to the control of the pH of the sample and washing solutions and the choice of washing solvents.

26. Factors requiring attention in SPE with silica gels
The elution solvent must overcome both the primary interactions of the analyte with the bonded phase and any secondary interactions with silanol groups.
The amount of fines (fine particles of silica gel) generated by different manufacturers’ cartridges varies. Ideally very little fines should elute from the column with the sample. Fine particles of silica gel can damage HPLC systems and interfere in derivatisation reactions used prior to analysis by GC.

27. Choice of SPE Device
Choosing the proper SPE device for your application depends on:
The nature of the sample and the analyte
Sample volume
Degree of contamination
Complexity of sample matrix
Quantity of compounds of Interest
Type and solvent strength of sample matrix.

28. Affinity Chromatography
Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner. Examples include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions.
Affinity chromatography offers high selectivity, capacity and efficient purification of proteins and related compounds. It has the advantage of utilizing a protein's biological structure or function for purification

29. IMAC (Immobilized Metal Affinity Chromatography)
Uses interactions between protein and chelated metal to separate phosphorelated proteins
Immunoaffinity
Quantitative determinination of antigens, high specificity, purification of specific proteins
Protein A / Protein G
Purification of immunoglobulins
Lectins
Selective extraction of Glycoproteins and other carbohydrate metabolite proteins.

30. Size exclusion chromatography (SEC)
SEC is a separation technique based on the molecular size of the components. Separation is achieved by the differential exclusion, from the pores of the packing material, of the sample molecules as they pass through a bed of porous particles.
Molecules of various sizes flow into the column, smaller molecules flow more slowly through the column because they penetrate deep into the pores, whereas large molecules flow quickly through the column because they do not enter the pores. Consequently, larger molecules elute from the column sooner and smaller molecules later, which effectively sorts the molecules by size.

31. Size exclusion chromatography (SEC)
Gel filtration chromatography
Gel permeation chromatography