Preperative HPLC.pptx

Basic of preparative HPLC with principle and case study.

1. PREPaRATIVE High performance liquid chromatography Presented By : - Atul Nagar Roll no 07 Under the guidance of Dr.(Mrs.) Purnima D. Hamrapurkar 1 Prin. K.M. Kundnani college of pharmacy Department of Pharmaceutical analysis

2. Contents :- INTRODUCTION PRINCIPLE INSTRUMENTATION Hyphenation techniques with preparative hplc :- HYPHENATION OF PREPARATIVE HPLC WITH Mass spectroscopy . HYPHENATION WITH counter-current chromatography. Hyphenation of preparative hplc with flash chromatography. 6. APPLICATIONS OF PREPARATIVE HPLC 7. REPORTED STUDIES using prep hplc 2

3. Introduction:- Chromatography is an important technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Michael Tswett is known as the father of chro matography , Tswett developed hís ideas in the early 1900’s about chromatography. To increase the throughput and separation power, the first preparative HPLC system was develop in 1970’s . The Preparative High Performance Liquid Chromatography is powerful technique used for the isolation and purification of variety of compounds. The aim of prep. HPLC experiment is to isolate the maximum amount of analyte of interest at a desired purity in short period of time. Prep. HPLC typically involves, working with high concentration and volume which is required for analytical purposes therefore detector selectivity and sensitivity are less important parameters. 3

4. PRINCIPLE Adsorption Similar to analytical HPLC The only main difference is that in that the sample goes through the fractional collector 4

5. Types of HPLC :- based on scale of operation. 5

6. DIFFERENCE BETWEEN ANALYTICAL AND PREPARATIVE HPLC:- 6

7. INSTRUMENTATION : The basic components of preparative HPLC instrument are shown in figure. A. Solvent reservoir B. Pump C. Preparative injector D. Preparative column E. Detector F. Diverter valve G. Fraction collector Figure : Schematic representation of components of Prep. HPLC 7

8. A. Solvent Reservior : Large capacity ( upto several gallons) reservoirs made up of glass or stainless steel are used in prep. HPLC. Material of construction varies with type of material. For biologically sensitive material, biocompatible material is used. B. Pump : P rep. HPLC requires high eluent flow rate and internal diameter of column used in prep. HPLC is usually larger. The required flow rate for prep. HPLC is usually between 10 to 100 ml/min (For larger columns, flow rate of 500 ml to 1 litre/min (may be required) For prep. HPLC. Analytical pumps are modified with respect to high pumping rates and large volume. The main modification that is required to work at flow rate of 10-100ml/min is a larger piston head with higher volume liquid filled chamber . 8

9. C . Preparative injector: Rheodyne injector should be able to inject the sample within the range of 0.1 to 100 ml of volume under high pressure (up to the 4000 psi). It is also referred as rotary sampling valve . D. Preparative column: The role of the column is very important in developing a reproducible prep. HPLC method. Preparative columns should be able to withstand the high inlet pressure, necessary to obtain the required flow rate. Particle size of a packing material used for column defines the performance of prep. column. As the preparative columns are wide sample distribution plate is placed for the distribution of the sample across the column. The distribution plate consists of a disc with a radial slots. 9

10. Packing of preparative column: Choice for the type of technique used for packing of preparative columns depends on the particle size of packing material, scale of the preparation and nature of the material to be separated. Dry packing and/or slurry packing is used for packing of preparative columns. 10

11. E. Detector : Prep. chromatographic detectors can have very limited specifications. They need not be sensitive as the sample size and the eluent solute concentrations are very large. Analytical detectors can be used for preparative purposes. In prep. HPLC sample should be diluted with large volume of mobile phase and then passed through the detector. F. Fraction collector : P rep. HPLC the diverter valve is used to divert the flow of sample either to waste or the desired part of the injected sample into a fraction container via the fraction collection needle. The preparative scale fraction collector requied flow rate up to 100 ml/min. Figure : Eluent flow rate 11

12. Hyphenation techniques with preparative hplc Prep. HPLC when coupled/ hyphenated with other analytical techniques, it offers several advantages in many ways. Prep. HPLC can be hyphenated with mass spectroscopy and other chromatographic techniques such as counter current and flash chromatography. Literature survey reveals that it is effectively used for purification and separation by combining it with mass spectroscopy, counter current chromatography as well as with flash chromatography. Reported studies of analysis making use of hyphenation of prep. HPLC are reviewed as follows, Preparative high-performance liquid chromatography– mass spectrometry (MS) for the high-throughput purification of combinatorial libraries. B. Combination of counter-current chromatography and preparative high- performance liquid chromatography to separate galactolipids from pumpkin. C. Preparative high pressure liquid chromatography-flash chromatography ( PuriFlash ). 12

13. Hyphenation techniques with preparative hplc Preparative high-performance liquid chromatography–mass spectrometry (MS) for the high-throughput purification of combinatorial libraries: - Prep. HPLC has been used for peptide and combinatorial chemistry. Fraction collection is achieved after UV detection Every UV- active compound can be collected above a certain threshold. The main advantage of prep. HPLC–MS is that, only the target molecule is selected by detecting the ion current of the desired target molecule . Figure : Set up of preparative HPLC-MS system 13

14. Advantages: 1. Online identification. 2. Ease of operation. 3. Non-critical fraction collector capacity. 4. There is one-injection, one-fraction purification procedure which solves many problems, normally encountered with large libraries. Disadvantages : If for any reason an existing product is not detected by MS, the sample is lost. 14

15. APPLICATIONS OF PREPARATIVE HPLC :- 1. Purification in medicinal or high-throughput chemistry. 2. Purification of compounds in natural product chemistry. 4. Recovery collection 3. Purification of by-products, formed during impurity analysis. 15 5. Automated fraction re-analysis: Automated fraction re-analysis consist of three steps: a. Pre-preparative analysis. b. Purification. c. Post preparative analysis.

16. Reported studies using preparative HPLC:- purification of saffron secondary metabolites picrocrocin and crocetin glycosyl esters. INTRODUCTION - Saffron is a very high-value spice obtained from dried Crocus sativus stigmas. It is mainly employed to provide colour and flavour to foods. The most important secondary metabolite in saffron are picrocrocin and glycosyl esters. Picrocrocin is a colourless bitter glycoside ,responsible for the bitter taste of saffron and precursor of safranal . 16 Sample Preparation - Saffron was ground, resuspended in 50% ethanol and stirred; the vegetal tissue residue was discarded after centrifugation. The hydroalcoholic extract obtained was vacuum concentrated, and the aqueous solution obtained was lyophilized and stored at -20°C until use. Samples of extract (from 10 to 200 mg ml-‘) were prepared by dissolving the lyophilized extract in water.

17. . Chromatographic Conditions 1. Mobile phase: methanol 45 % and acetonitrile 55 % 2. Column: C18 (25 cm x 0.46 cm I.D. and 25 cm x 2.12 cm I.D.) 3. Flow rate: 42 ml/min. 4. Detector: UV-Vis spectrophotometric detector ( SPDdAV ) 5. Pump : LC-8A pumps, 6 . Fraction integrator - FCV-1OOB fraction collector and a CR4A Chromatopac integrator 17 Method development Preparative HPLC analyses were performed on a Shimadzu HPLC system equipped with two LC-8A pumps, injectors for analytical and preparative runs, a UV-Vis spectrophotometric detector ( SPDdAV ) with analytical and preparative flow cells, an FCV-1OOB fraction collector and a CR4A Chromatopac integrator. Columns used were 25 cm x 0.46 cm I.D. and 25 cm x 2.12 cm I.D. ( Supelco ) for the analytical-scale (referred to below as the small-scale column) and the preparative-scale assays, respectively, both packed with Supelcosil PL C-18 (12 pm particle size). The standard mobile phase used for preparative separation of picrocrocin and pigments was isocratic 45% methanol, plus a 6-s pulse of 90% acetonitrile 3 min after starting the run.

18. The sample solution was manually injected with a syringe. Flow-rates were 2 and 42 ml min-’ and sample sizes 20 ~1 and 2 ml for small-scale and preparative assays, respectively. Picrocrocin was detected at 250 nm and pigments at 440 nm for small-scale assays. As crocin elution could also be detected at 250 nm, for preparative runs all compounds were monitored at 250 nm. All separations were performed at room temperature. The identification of saffron secondary metabolites was made by HPLC using commercial crocin and previously purified picrocrocin as standards. Picrocrocin and crocin were quantified using their molar absorptivities in water. 18

19. Result and Conclusion : The highest yield of picrocrocin with the 80% purity, obtained by preparative HPLC method development. In conclusion, a simple, efficient and reproducible method for the purification picrocrocin and saffron pigments by preparative HPLC has been developed. In addition, the results reported in the present work are a contribution to the possible industrial use of picrocrocin and saffron pigments separately. 19

20. REFERENCE Bharti G. Jadhav, Amruta M. Jadhav ” A Comprehensive Review for the Learners and Users: Preparative High Performance Liquid Chromatography” , Internat i iona l l Journa l l of CHEMICAL AND PHARMACEUTICAL ANALYSIS , 1 July 2014 Castellar M, Montijano H, et al, Journal of chromatography, 1993, (648): 187-190. https://www.slideshare.net/BhartiJadhav1/prep-hplc-1 20

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