Chromatography

1. Chromatography

2. Chromatography Definition: Chromatography is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium. Components: - mobile phase: a solvent that flows through the supporting medium - stationary phase: a layer or coating on the supporting medium that interacts with the analytes - supporting medium: a solid surface on which the stationary phase is bound or coated

3. The analytes interacting most strongly with the stationary phase will take longer to pass through the system than those with weaker interactions. These interactions are usually chemical in nature, but in some cases physical interactions can also be used.

4. Types of Chromatography: Chromatography can be classified based on the type of mobile phase, stationary phase and support material

5. Types of Chromatography 1.) The primary division of chromatographic techniques is based on the type of mobile phase used in the system: Type of ChromatographyType of Mobile Phase Gas chromatography (GC) gas Liquid chromatograph (LC) liquid 2.) Further divisions can be made based on the type of stationary phase used in the system: Gas Chromatography Name of GC MethodType of Stationary Phase Gas-solid chromatography solid, underivatized support Gas-liquid chromatography liquid-coated support Bonded-phase gas chromatography chemically-derivatized support

6. Types of Chromatography Liquid Chromatography Name of LC MethodType of Stationary Phase Adsorption chromatography solid, underivatized support Partition chromatography liquid-coated or derivatized support Ion-exchange chromatography support containing fixed charges Size exclusion chromatography porous support Affinity chromatography support with immobilized ligand

7. Learn the basic concept of High Pressure Liquid Chromatography 01. Introduction https://youtu.be/IUwRWn9pEdg 02. The Mobile Phase https://youtu.be/pmHtGDdagJU 03. The Stationary Phase https://youtu.be/MYSBOxbnuAw 04. Normal Phase HPLC vs Reverse Phase HPLC https://youtu.be/MLoitPJQH3g 05. HPLC Isocratic vs Gradient analysis http://youtu.be/tAcfJPveWwM 06. HPLC - UV-VIS detection of analytes https://youtu.be/sfxEj_MxBcs 07. HPLC - How to read a chromatogram? https://youtu.be/qXmSb6Xwr5k

9. Principles of Chromatography https://www.youtube.com/watch?v=_CWtWeEN7is https://www.youtube.com/watch?v=0m8bWKHmRMM

10. 3.) Chromatographic techniques may also be classified based on the type of support material used in the system: Packed bed (column) chromatography Open tubular (capillary) chromatography Open bed (planar) chromatography

11. 4.) Chromatographic techniques could also be classified based on the type or the purpose of the analysis or scale of operation: A- Analytical Chromatography B- Preparative Chromatography

12. Theory of Chromatography 1.) Typical response obtained by chromatography (i.e., a chromatogram): chromatogram - concentration versus elution time Wh Wb Inject Where: tR = retention time tM= void time Wb = baseline width of the peak in time units Wh = half-height width of the peak in time units

13. Note: The separation of solutes in chromatography depends on two factors: (a) a difference in the retention of solutes (i.e., a difference in their time or volume of elution (b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation system) A similar plot can be made in terms of elution volume instead of elution time. If volumes are used, the volume of the mobile phase that it takes to elute a peak off of the column is referred to as the retention volume (VR) and the amount of mobile phase that it takes to elute a non-retained component is referred to as the void volume (VM). Peak width & peak position determine separation of peaks

14. Efficiency: Efficiency is related experimentally to a solute’s peak width. - an efficient system will produce narrow peaks - narrow peaks  smaller difference in interactions in order to separate two solutes Efficiency is related theoretically to the various kinetic processes that are involved in solute retention and transport in the column - determine the width or standard deviation (s) of peaks Wh Dependent on the amount of time that a solute spends in the column (k’ or tR)

15. Number of theoretical plates (N): compare efficiencies of a system for solutes that have different retention times N = (tR/s)2 or for a Gaussian shaped peak N = 16 (tR/Wb)2 N = 5.54 (tR/Wh)2 The larger the value of N is for a column, the better the column will be able to separate two compounds. - the better the ability to resolve solutes that have small differences in retention - N is independent of solute retention - N is dependent on the length of the column

16. Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of columns with different lengths: H = L/N where: L = column length N = number of theoretical plates for the column Note: H simply gives the length of the column that corresponds to one theoretical plate H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size, etc.) to the kinetic processes that give rise to peak broadening:

17. Why Do Bands Spread? a. Eddy diffusion b. Mobile phase mass transfer c. Stagnant mobile phase mass transfer d. Stationary phase mass transfer e. Longitudinal diffusion

18. a.) Eddy diffusion– a process that leads to peak (band) broadening due to the presence of multiple flow paths through a packed column. As solute molecules travel through the column, some arrive at the end sooner then others simply due to the different path traveled around the support particles in the column that result in different travel distances. Longer path arrives at end of column after (1).

19. b.) Mobile phase mass transfer– a process of peak broadening caused by the presence of different flow profile within channels or between particles of the support in the column. A solute in the center of the channel moves more quickly than solute at the edges, it will tend to reach the end of the channel first leading to band-broadening The degree of band-broadening due to eddy diffusion and mobile phase mass transfer depends mainly on: 1) the size of the packing material 2) the diffusion rate of the solute

20. c.) Stagnant mobile phase mass transfer– band-broadening due to differences in the rate of diffusion of the solute molecules between the mobile phase outside the pores of the support (flowing mobile phase) to the mobile phase within the pores of the support (stagnant mobile phase). Since a solute does not travel down the column when it is in the stagnant mobile phase, it spends a longer time in the column than solute that remains in the flowing mobile phase. The degree of band-broadening due to stagnant mobile phase mass transfer depends on: 1) the size, shape and pore structure of the packing material 2) the diffusion and retention of the solute 3) the flow-rate of the solute through the column

21. d.) Stationary phase mass transfer– band-broadening due to the movement of solute between the stagnant phase and the stationary phase. Since different solute molecules spend different lengths of time in the stationary phase, they also spend different amounts of time on the column, giving rise to band-broadening. The degree of band-broadening due to stationary phase mass transfer depends on: 1) the retention and diffusion of the solute 2) the flow-rate of the solute through the column 3) the kinetics of interaction between the solute and the stationary phase

22. e.) Longitudinal diffusion– band-broadening due to the diffusion of the solute along the length of the column in the flowing mobile phase. The degree of band-broadening due to longitudinal diffusion depends on: 1) the diffusion of the solute 2) the flow-rate of the solute through the column

23. resolution(RS) – resolution between two peaks is a second measure of how well two peaks are separated: RS = where: tr1, Wb1 = retention time and baseline width for the first eluting peak tr2, Wb2 = retention time and baseline width for the second eluting peak tr2 – tr1 (Wb2 + Wb1)/2 Rs is preferred over a since both retention (tr) and column efficiency (Wb) are considered in defining peak separation. Rs$1.5 represents baseline resolution, or complete separation of two neighboring solutes  ideal case. Rs$ 1.0 considered adequate for most separations.

24. Brief Description of Column Chromatography https://www.youtube.com/watch?v=k9A-EXTbqZY

25. HPLC

26. HPLC Analysis | Working Principle and Method of this chromatography - HPLC analysis is one of the types of chromatography used to isolate and analyze mixtures. - HPLC in full form is “High-pressure liquid chromatography.” - But also due to its efficiency in the analysis of compounds, it is regarded as High-performance liquid chromatography. - On the humorous side, some even call it as High patience liquid chromatography based on long time duration needed to run it.

27. HPLC chromatography is one of the widely used techniques in the fields of; a. Clinical research, b. Biochemical research, c. Industrial quality control, etc. Applications of HPLC include detection, analysis, determination, quantification, derivation of molecules from mixtures (prep HPLC) of biological, plant and medical importance. Before going into the details of HPLC theory, principle and further HPLC tutorial, let’s see how it evolved and why?

28. Why HPLC? Before the discovery of chromatography, techniques like gravimetric analysis, photometry, colorimetry (UV, visible detection), titrimetry (acid-base detection), etc.. were sole methods available for analysis. Even the requirements of analysis for research was simple i.e. there was no necessity for analysis of complex molecules, similar molecules (i.e., molecules with the same chemical and physical properties). But as research advanced there was the requirement to analyze all the molecules in a given sample for better detection of the problem (in the clinic), impurities and also deficiencies in industry and research. This was not possible with a single technique like photometric, titrimetric, etc. due to greater physical and chemical similarity in molecules of a sample like phyto-constituents, amino acids, neurotransmitters, etc.. With this limitation in mind, further efforts by scientists led to the development of HPLC chromatography with further improvements in speed and efficiency of analysis. Thus HPLC principle was discovered to analyze like compounds or similar compounds at a faster rate with better efficiency. HPLC instrument has a injection system, a pump, column, detectors and a recorder.

29. Collection of 7 videos about HPLC should be considered: https://www.youtube.com/watch?v=IUwRWn9pEdg&list=PLetrcQ_JUQSmK4yEiMDyMoiaaZYEeQPDy

30. HPLC Analysis Principle The principle involved in HPLC testing is the separation of compounds in a mixture more efficiently and also quickly than that of traditional column chromatography. Segregation of compounds is due to their relative differences in travel through the column on the application of pressure exerted through the mobile phase or carrying liquid. The compounds of the mixture travel at different rates due to their relative affinities with the solvent and stationary phase. Compound with a higher affinity towards the stationary phase of the column moves slowly and vice-versa. The above principle is similar to that of column chromatography but in HPLC, The separation is more efficient due to greater surface area achieved due to a tiny particle size of stationary phase in comparison to that used in column chromatography. To minimize this obstacle, the high pressure is applied to the flow of HPLC mobile phase through the column by use of pumps.

31. The HPLC procedure is as follows All the chemicals and reagents used for the process should be of HPLC grade for efficient and smooth analysis. ♦ The mixture required to be evaluated is injected by HPLC injection into a stream of mobile phase which is flowing at a defined pressure. ♦ The injected mixture now does flow over the stationary phase inside the column under the influence of pressure along with the mobile phase. ♦ During this flow based on the affinity of individual compounds in the mixture towards stationary and mobile phase, some compounds get eluted first out of the column and others later. ♦ Outside the column they are sent into a detector where individual compounds are detected and recorded in a computer-installed chromatography software. ♦ The recordings (preferably in the form of quantitative peaks) are compared with those of standard compound’s HPLC values, and the individual compounds are identified.

32. For an overview of HPLC system and operation see the video tutorial below https://www.youtube.com/watch?v=I-CdTU5X4HA https://www.youtube.com/watch?time_continue=329&v=kz_egMtdnL4 UPLC https://www.youtube.com/watch?v=Qf6060arkqM Please open it in your mobile phone and check each video

33. Types of HPLC • HPLC analysis is of differentiated based on: • The stationary phase in the column used:Based on the nature of stationary phase used it can be either normal phase or reverse phase HPLC. • Normal phase chromatography: Here the stationary phase of the column is made of polar compounds like silica gel, alumina, etc.. The polar compounds or molecules in the sample under analysis have higher affinity to the stationary phase, and so they are retained longer in the column than non-polar ones. Hence non-polar compounds are eluted first under the affinity to non-polar mobile phase while polar ones are eluted later.

34. 2. Reverse phase HPLC: Here exactly the opposite of normal phase happens. The stationary phase is made of non-polar compounds like C18, C8 type of organic compounds. The mobile phase used is polar. So compounds of high polarity or eluted first while those of low polarity or no polarity are eluted last. Most of the applications in HPLC require the evaluation of drugs, biochemical molecules and other substances used by humans and they are polar (water soluble) in nature. So, reverse phase HPLC is widely used.

35. b) Based on purpose of use: Here HPLC is used for either 1. Analysis Mode: The procedure is done to estimate different types of molecules and their individual quantities in the mixture using the help of a detector. 2. Preparative mode: Here the intention of the process is to separate large amounts of a particular molecule from a mixture. The molecule or substance eluted is of the highest purity. The column size, the sample size is comparatively large than that of analytical mode. HPLC advantages and disadvantages

36. It will be interesting to know HPLC is one of the few appliances in research which is used at the expense of its considerable disadvantages. This is because of the advantages which only it provides and not replaceable by other equipment. ♣ Advantages of HPLC: • It includes both aspects of analysis, i.e., qualitative and quantitative analysis. • HPLC method evaluates almost all the molecules of the same family. • For example, in one single run, all the monoamines like dopamine, epinephrine, serotonin can be estimated. A single run for steroids in one sample gives the data of all the steroids in that sample. • Molecules with small differences in absorption wavelengths can be detected well due to their differences in separation time. I.e., one which travels faster is measured before the other which is measured later. This is the prime advantages if HPLC which makes it non-replaceable.

37. • Substances in very low concentration like Nano and picograms can be detected due to the sensitivity of HPLC detectors used like the electrochemical detector, fluorescence detector, etc. • Due to its high separation efficiency, the quality of substance obtained by preparative mode or technique (prep HPLC) is of high purity.

38. ♣ Disadvantages of HPLC: • It’s an expensive method as it requires costly HPLC instrumentation, columns and also uses of the highest grade of purity solvents, buffers, chemicals, etc. termed as HPLC grade. • Working on HPLC requires heavy processing before estimation like mixing, homogenization, filtration, degassing, derivatization, etc. These techniques are also to be performed with proper care to avoid problems in estimation. • The systems operation requires prior HPLC training and active HPLC troubleshooting skills. So prior practice is essential to run these chromatography systems. • HPLC Data obtained is non-homogenous and is never without any noise (fluctuation) and errors during estimation.

39. • It’s time-consuming, and you must have a good amount of patience. • Alteration in temperature and presence of dust in chromatography lab can greatly vary the result output. So strict maintenance of experimental conditions is required throughout the process.

40. Thank you 🙂

41. HPTLC-High performance thin layer chromatography HPTLC introduction HPTLC is an abbreviation for High-Performance Thin layer Chromatography or High-Pressure Thin Layer Chromatography. This is a sophisticated advancement of Thin-Layer chromatography (TLC). It has added advantages like better resolution, faster development of spots and also easy detection and quantification of separated compounds. The advancement concerning better separation and resolution and faster resolution is due to 1) Use of ready-made HPTLC plates with optimized absorbent layers having smaller particles size with uniform particle size distribution as the stationary phase. 2) The use of pressure enables for faster development of chromatograms in even complex mixtures. The HPTLC system is further automated to a greater extent than its precursor TLC as. a) Auto sampling for application of samples on the plates avoiding manual sampling errors. b) Online mixing of solvents for either gradient mode (i.e., a mixture of solvents in a predefined ratio) or isocratic mode ( single solvent) required during chromatogram development.

42. c) Auto-detection of compounds from chromatogram using built-in UV and fluorescent detectors detector system. d) Recording and storage of data from chromatograms utilizing an HPTCL data software in a computer. Advantages of HPTLC over TLC: ♦ Samples in minute quantities like in nano-gram range can be detected using HPTLC. ♦ Handling and human errors are minimum due to automation. ♦ Better accuracy and sensitivity than TLC. Disadvantages: ♣ The system is many folds expensive than TLC. ♣ Bulky instrumentation and large space requirement. ♣ Requires stringent condition of operation like dust free environment and temperature controlled conditions. ♣ A technically skilled person with the knowledge to run the system is needed. Application of HPTLC: 1) For detection and analysis of components of phytochemistry, medicinal chemistry & organic chemistry. 2) In TLC one can identify the elements of an extract. Whereas using this method, one can even estimate it concentration. 2) Compounds having a complicated structure or those available in very scarce quantities can be analyzed.