INTRODUCTION:

Chromatography is a method of separation, that distributes a mixture of components in the two separate phases, one stationary phase and another a mobile phase moving in a distinct direction leading to the separation. The principle of separation can be either difference in their partition coefficients or adsorption coefficients.^[1]

Chromatography is one of the major separation techniques used in the pharmaceutical and chemical industries. Chromatography can be broadly divided into two types based on its purpose of use.

Analytical chromatography:

The objective of this technique is to perform the qualitative and quantitative analysis of the different components present in a given sample.

Preparative chromatography:

The objective of this technique is to purify a sample and to isolate the purified analytes for the further steps.^[2]

Column chromatography was the method used in the industries for the purification purpose and it offers many advantages like being inexpensive, simple packing and easy operation. But it also has certain drawbacks like being time consuming and low resolution. It was engaged in numerous areas for the purification and isolation of components. Column chromatography is a particularly lengthy stage and can promptly turn into the hurdle for any process lab. This pressurized the demand for the development of a new and rapid liquid chromatographic technique, where in the eluent flows down the column rapidly with the help of positive pressure.^[3]

In 1978, an American organic chemist, Clark Still had developed a purification technique; in which the pressure was applied on the top of the column in liquid chromatography. This led to increase in the rate of solvent elution and significantly reduced the run time. By using this technique, epimers of benzyl alcohol were separated.^[4]

Principle involved in flash chromatography:

Flash chromatography is based on the principle of adsorption chromatography. The components of a mixture are separated based on the differences in their affinity towards stationary phase and mobile phase. The component which has more affinity towards the stationary phase moves slower and the component which has more affinity towards the mobile phase moves faster. But the special characteristic of flash chromatography is the use of positive pressure to pump the mobile phase across the column, resulting in shorter run time.^[5]

Advantages of flash chromatography^[6]

Flash chromatography offers various advantages.

· It is a rapid method. The elution completes in a flash.

· It is a low cost technique.

· Components of a mixture offer more resolution.

· The components can be separated and collected up to few grams.

· This can be used in low scale and also in high scale.

· The method can be automated.

· Compounds which often degrade in the column can be eluted in purified state in flash chromatography, as the contact time is less.

· This technique is eco friendly, as the solvent usage is also less.

· The column can be reprocessed numerous times and save the time for preparing the column.

· The hazardous health effects on the operating personnel are less, due to less time of operation^[5,6]

Instrumentation of flash chromatography^{[7, 8]}

Flash chromatograph includes different parts which are similar to preparative HPLC. It is mainly distinguished by the presence of flash pump and fractional collectors.

· Pump Systems

· Sample Injection Systems

· Pre-columns

· Columns

· Fractional Collectors

· Detectors

· Recorder and amplifier

Pump systems:

Different types of pumps like vaccum pump, peristaltic pump are used. They pump air or nitrogen into the column. The speed of the gas can be controlled using controller and manager units.

Sample Injection Systems:

The sample can be loaded on to the column using the injector port and injector valve.

Pre-columns:

These short columns are placed before any column so as to increase the life of column. They work by reducing the dead volume and by trapping the contaminants.

Columns:

Different columns can be used made of glass or plastic. It is also not uncommon to use prepackaged columns. These columns are highly economical as they can be reused. These columns can be filled either with dry silica or silica slurry. Previously only normal phases were used. Now even the reverse phase chromatography can be performed in the flash chromatographic set up, by using a modified adsorbent (like ODS).

Fractional collector:

Fractional collector is a device used to collect the eluate of the chromatographic system. The different fractions of the eluate can be collected by attaching it to the column outlet. Monitoring of these separations can be monitored online or offline. The quantity of the gathered portions largely depends on the internal diameter of the column, time required for separation and the flow rate ^{[9, 10]}.

Detectors:

Different detectors can be used for the identification and quantification of different components collected. Generally, UV-spectrophotometer or differential refractometers are used.

UV-Spectrophotometer:

It works on the principle of absorption spectroscopy. The analytes are identified and quantified based on the wavelength and intensity of absorbed radiation.

Differential Refractometer:

It works on the principle of Refractometry. The refractive index of pure solvent differs with the type and amount of analyte present in a solution.

Procedure of flash chromatography

The different steps for flash chromatography can be recapitulated in the following steps:

Ø Selection of following:

· The eluent

· Adsorbent

· Column properties

· Different chromatographic conditions

Ø Preparation of column

· Dry packing method

· Wet packing method

Ø Introduction of sample

Ø Running the column

· Isocratic elution

· Linear gradient elution

· Step gradient elution

Ø Sample collection

Ø Analyte detection

Selection of Eluent:

The selection of mobile phase depends on the properties of the analytes to be separated and collected. The composition of mobile phase can be single or a mixture of solvents. The different solvents which can be used in flash chromatography are n-hexane, 2, 2, 4-trimethyl pentane, cyclohexane, 1, 1, 2-trichloromethane, toluene dichloromethane, ethyl acetate, methyl-t-butyl ether, acetone, tetrahydrofuran, acetonitrile, isopropanol, ethanol, methanol and water. The pH of the mobile phase can also be adjusted with the help of different buffers.

Selection of adsorbent:

The stationary phase to be used in the chromatography depends on the physico-chemical nature of the components to be separated and the presence of different contaminants. The adsorbent can be silica, modified silica or alumina.

Selection of column properties:

The dimensions of the column depend on the quantity of the sample required to analyze, amount of the adsorbent required for the separation, number and nature of impurities and affinity of the components towards the adsorbent used.

Selection of chromatographic conditions:

Different conditions like flow rate, column temperature and the pressure of pumped gas depend on the various properties of the adsorbent, eluent and the analytes.

Preparation of column:

The bottom portion of column is packed with cotton wool or the asbestos pad, and then it is packed with adsorbent. This packing can be done in two ways.

Dry packing method:

The adsorbent is packed in the dry form and then the solvent is poured over it. Trapping of air bubbles can be prevented by tapping the column prior to transferring the solvent in to it.

Wet packing method:

The slurry of the adsorbent is prepared first and then it is transferred in to the column.

Now a days, prepacked columns or cartridges are being used. These columns can be reused several times by simple column washing.

Introduction of sample:

The sample solution is introduced in to the column through injector port and it is then loaded on to the column.

Running the column^[11]:

Like conventional chromatography, different elution techniques can be used in the flash chromatography.

· Isocratic elution:

The composition of the eluent system remains same for the entire elution period.

· Linear gradient elution:

The proportion of stronger solvent is gradually increased over time in the eluent system.

· Step Gradient Elution:

There is sudden increase in the proportion of stronger eluent at definite time intervals.

Sample Collection:

The various fractions of the eluate are collected at defined time intervals with the help of fractional collector.

Analyte detection:

The different fractions obtained from the fractional collector are then analyzed using UV detector or refractometer. Depending on the results obtained the samples in the fractions are identified.

Association between TLC and flash chromatography^[12]:

During the method development of flash chromatography, the sample mixture is first subjected to TLC to optimize different conditions. As TLC is a rapid and inexpensive technique, many trials can be done and it also gives an idea regarding solvent system composition, amount of adsorbent to be used.

· The eluent system which has an R_fequal to 0.15 or 0.2 in TLC for the analyte is then selected. The retention factor should be low because the surface area of adsorbent in TLC is almost double the silica gel surface area in a column chromatography.

· It is used to estimate column volume. Column volume (CV) is the volume inside the column which is not occupied by any media. It includes internal porosity, interstitial volume and the empty volume in the column. This column volume indicates the loading capacity and separation efficiency of a column.CV is the inverse value of retention factor. The differences between the CV values of components give an indication of their separation efficiency in column chromatography.

Consideration between HPLC and flash chromatography:^[13,14]

High pressure chromatography is a widely used technique in various laboratories and industries. The main application of HPLC includes separation of minute quantities of sample, their identification and quantification. This instrument is also highly expensive for installation and operation.

· Flash chromatography is a technique widely used for purification of desired sample. Its application is to isolate purified samples in large quantity. It is mainly concerned with the separation of large quantity of samples within less time.

· Flash chromatography is preferred over the frequently used high cost preparative HPLC, if the components to be separated are in high concentration.

Applications^[15]:

Flash chromatography is used in various fields for separation and collection of individual compounds.

1. Flash chromatography is exploited for severance of associated Organic Compounds.

2. Flash chromatography is utilized for High Speed Flash Fractionation of Natural Products.

3. Flash chromatography is used to decontaminate, accumulate and recognize a variety of aromatic constituents in a semi-synthetic extort.

4. Flash chromatography is employed in refinement of mixture of peptides, antibiotics.

5. Flash chromatography is utilized in isolation of strongly linked drug intermediates.

6. Flash chromatography is used during the process of drug discovery.

7. Flash chromatography is used in agricultural chemistry.

8. Flash chromatography is exploited during refinement and processing of petroleum products.

CONCLUSION:

Flash Chromatography is an unsophisticated, rapid, cost effective preparative liquid chromatography with minimal instrumentation requirements. It is an excellent preparative chromatographic tool that is still routinely used in many laboratories, mainly during drug discovery process. This technique offers many advantages like easy installation and operation, usage of less quantity of solvents, purification of large amount of samples, consumption of less time. Hence, flash chromatography is an affordable chromatographic technique which can be used when moderate resolution is required.

REFERENCES:

1. Skoog, instrumental analysis, Indian edition, pg no-836.

2. http://analyteguru.com/analytical-and-preparative-liquid-chromatography-what-are-the-main-differences/

3. Swathi G, Srividya A, Ajitha A, Uma Maheswar Rao V. Review on flash chromatography. Available from URL: www.wjpps.com/download/article/1438354107.pdf

4. Clark still W, Michael Kahn, and Abhijit Mitra, Rapid Chromatographic Technique for Preparative Separations with Moderate Resolution. Available from URL : https://www.sas.upenn.edu/~marisa/documents/fcolumn.pdf

5. https://en.wikipedia.org/wiki/W_Clark_Still”

6. http://medchemblog.blogspot.com/2009/10/flash-chromatography.html

7. https://www.pharmatutor.org/articles/flash-chromatography-area-applications?page=1

8. Hostettmann K and Terreaux C medium pressure liquid chromatography, academic press. pp: 3296-3303

9. C. F. Poole, Flash chromatography ,academic press, pp : 2808-2814

10. http://spectrumlabs.com/chrom/cf2.html

11. http://www.flash-purification.com/how-to-choose-between-linear-gradients-and-step-gradients-for-flash-chromatography/

12. http://www.flash-purification.com/so-what-exactly-is-a-column-volume-in-flash-column-chromatography-and-how-is-it-determined/

13. Ramesh Dattatraya Bhusal, Deepali Mahir Nahar, Prashant Bhimrao Dalvi. Review on: flash column chromatography. Available at URL: https://zenodo.org/record/1006789/files/16.pdf.

14. http://www.biotage.com/news/find-out-why-the-era-of-regular-flash-has-come-to-an-end

15. https://www.slideshare.net/shishirkawde/flash-chromatography

Received on 09.08.2018 Modified on 15.09.2018

Asian J. Research Chem. 2018; 11(5): 815-818.

DOI: 10.5958/0974-4150.2018.00144.X