DESC: SLURRY PACKING METHOD AND SYSTEM FOR HPLC COLUMNS
FIELD OF INVENTION
[001] The embodiments herein generally relate to the field of media packing system and media packing methods for columns, more specifically, it relates to an improved method and system for slurry packing of columns of aspect ratio 1.0-120 for use as high pressure liquid chromatography (HPLC) columns.
BACKGROUND OF THE INVENTION
[002] Liquid chromatography is now one of the most powerful tools in analytical chemistry. It has the ability to separate, identify, and quantitate the compounds that are present in any natural, biological, synthetic, therapeutic samples that can be dissolved in a liquid. The analysis of separated entities provides information about related constituents and bespeaks about the purity of substance once quantified. The device required for enabling this kind of separation, identification, assaying the purity of substances is known as analytical column in the realm of liquid chromatography. These analytical columns carry a special packing media of very low micron size particles, (known as stationary phase) which are inherently surface active, exerting physical affinity secondary force to the extent determined by the physicochemical characteristics of the entities (known as analyte) carried by passing at high pressure a liquid (known as mobile phase) through the column for separation.
[003] All the currently existing column technologies/products are predominantly based on the Slurry Packing and Dynamic Axial Compression methods or sometimes by cross breading the principles beneficial that go into the packing of effective columns which represents a significant part of world wide sales, today. The slurry method normally gives the best column packing, but is also a more difficult technique to master.
[004] In slurry packing method, the column is attached to a slurry reservoir in the flow path while the other end of a column is closed by an end fitting. The column chamber is packed by a valve in the flow path. However, the reservoir and the column are filled with the slurry of a liquid and the packing medium and this suspension is pumped into the column tube at suitable pressure subject to column aspect ratios and type for designated application interests. The miscible packing solvent, which may or may not be different from the slurry suspension solvent, drives the suspended media particles at certain flow rate. The particulate medium accumulates and gradually, a bed of packing medium builds up until it is full, well retained in the column tube modular of different lengths. Whereas, the packing solvent percolates through the bed of packing medium consolidates the packed bed as it builds and leaves the column tube.
[005] When packing from the top, the accumulated bed is compressed by the controlled pressure from the pumped liquid and by its own weight as upper part of the bed. In fact the absolute packing pressure values or profiles for a given column and media cannot be specified because consolidation takes place under the influence of the seepage force, which is the reaction of the bed to the pressure gradient required to maintain flow rate of the stream of liquid percolating through the bed. A common procedure is to continue pumping until the column is full, whereupon the pump usually cuts out spontaneously. As the liquid flow pressure is relieved, the packed bed then relaxes to fill the column with a decompression profile over its length such that the inherent variations subtle in the packed medium become unintelligible sometimes. However, after packing, the column is removed from the packing station and the inlet end is closed with an end fitting.
[006] Analytical columns precision and reproducibility of the analysis always depends not only on the method developed for analysis but also on the efficiency of the column processed using the slurry packing method. Although HPLC columns packing process is known, the state-of-art vis-à-vis skill levels are custom tuned and varies between each individual manufacturer in reaching the end-user in terms of performance. For column efficiency, the requirement is that the packed column has to be continuously, uniformly and entirely filled with the particulate medium under an appropriate degree of pressure. Under circumstance a host of design, processing and interrelated control parameters for a given medium in a given column requires processing method adjustment upon test trials and is time consuming.
[007] Analytical columns based on mixed bed strategy have been packed using the packing system and method. These columns offer reduction in the retention time (RT) of interest in industries related to proteomics and biosimilars for which attempts are made by several other workers to this effect using conventional analytical columns and columns as per pharmacopeial analytical methods. As combinations of traditional columns induce column mismatches and irregularities in such chromatographic analysis, mixed bed columns is one of the couple of innovative strategies.
[008] Semi-preparative or preparative HPLC columns could be packed by standard slurry packing method. Partially intriguing although and the stigma known is that the slurry packing method tends the bed of wide bore columns to collapse in places leading to pools of free liquid at the column inlet, convectional back mixing, column instability and inefficiency. However, to alleviate these effects detrimental, axial compression of the slurry in a direction parallel to the walls of the column or radial compression in the radial direction is adopted.
[009] It is known that this slurry packing method has the disadvantage in that the bed of the packing medium produced is radially heterogeneous, denser close too the wall than it is in the core region. Consequently, during testing process, the mobile phase moving faster in the core region than in the wall region causes the band to warp.
[0010] Further, it may be seen that the packing process for a given column configuration and media may not be specified because different columns may generate different back pressure determined by flow system length, diameter, number of pipe bends, slurry concentration variations, media particle and pore sizes, viscosity changes, induced vibrations, bed support type, limit pressures, temperature induced column expansion.
[0011] Therefore, there is a need to establish an efficient slurry packing method which is not only suitable for analytical, wide pore size, mixed bed, semi-preparative and preparative columns, but also in narrow bore columns that results in better performance characteristics.
[0012] Further there is a need in obtaining columns of all aspect ratios in the range (1-120) not only with good performance properties but also have an enhanced production capability of commercial significance.
OBJECTS OF THE INVENTION
[0013] Some of the objects of the present disclosure are described herein below:
[0014] The main object of the present invention is to provide a simple and efficient slurry packing method for packing of chromatography columns suitable for analytical columns, wide-pore columns, mixed-bed columns, semi-preparatory, preparatory and narrow bore columns.
[0015] Another object of the present invention is to provide a slurry packing method in quick time with enhanced production capability of the packing system.
[0016] Yet another object of the present invention is to provide an effective slurry packing method with better retentivity and resolution to detect additional peaks, better peak shape, lower pressure and temperature sensitivity of retention time or theoretical plates and provide realistic assays.
[0017] Still another object of the present invention is to provide a slurry packing method with efficient leak proof mechanism.
[0018] Another object of the present invention is to provide an evenly distributed and packed stationary phase in slurry packing method.
[0019] The other objects and advantages of the present invention will be apparent from the following description when read in conjunction with the accompanying drawing which is incorporated for illustration of preferred embodiments of the present invention and are not intended to limit the scope thereof.
SUMMARY OF THE INVENTION
[0020] In view of the foregoing, an embodiment herein provides an efficient method for slurry packing of chromatography columns, which is suitable for analytical columns, wide pore columns, mixed-bed columns, semi-preparatory, preparatory columns and narrow bore columns.
[0021] According to an embodiment, a method for achieving balanced density slurry-binary solvent is provided. Accordingly, the slurry-binary solvent is prepared by dispersing or suspending chromatographic octadecylsilyl media typified by particle sizes in the range of 1.5µ-15µ of pore sizes (40A -400A) and media having other bonded phases (C-18, C-8, C-4, -CN, -NH2, -C6H5, etc) in about 1g-500g in a binary mixture of solvents selected from a group comprising tetrabromoethane, CCl4, CHCl3 or aromatic (BrC6H5) halo hydrocarbon with viscous liquid, wherein the viscous liquid includes polyethylene glycol, cyclohexanol, ethanol and so on. The slurry-binary solvent having a good settling time lag is ascertained to have good density slurry-solvent density balance.
[0022] According to an embodiment, the ratio of halo hydrocarbon of appropriate densities and their volume proportions with a viscous liquid like polyethyleneglycol, cyclohexanol for suitable viscosity characteristics is preferably in the range of 30% - 70%. Additionally, static charge build up is minimized by an alkali metal addition in one of the solvent used, if necessary. The slurry, formed using the present solvent dispenser for controlled performance characteristics and enhanced production factor, is homogenized, ultrasonicated cold and the suspended particles monitored for minimum settling time of in between <30mins to <60mins depending on the aspect ratios. The slurry characterized to achieve a densely dispersed and uniform bed in columns is filled into the quick connect slurry reservoir and also into the column attached to the slurry reservoir.
[0023] According to an embodiment, packing solvent comprises of low density alcohols and water, wherein the alcohols includes methanol, isopropyl alcohol or combination thereof, wherein the packing solvent is stored in 5 to 20 liters reservoirs.
[0024] According to an embodiment, a system for slurry packing is disclosed. Accordingly, the system comprises of an air compressor, Haskel pump and accommodative manifold with snubber for hydrodynamic flow and pressure stabilization, pressure gauge, pressure valve regulator, an outlet adapter having spray distributor that enables uniform flow in the slurry reservoir to which modular columns are attached for packing.
[0025] According to an embodiment, a method of slurry packing columns including controlling air pressure and leak proof in the packing system is provided, wherein the method comprises the step of measuring pressure change [dp] due to the compressor pressure cycle, and determining characteristic time rated pressure difference [dp/dt] determined by the line volume fluidically connected to the pump. Whereas dp/dt monitored is < 1 indicative of no leak criterion and that dp/dt has minimal effect during priming of the pump and any pressure change is minimally communicated to the bed build up while packing in the column volume.
[0026] In a further embodiment, drawing packing solvent into the pump at the hydrodynamic volume in the range about 5ml to about 20ml, keeping the flow rates of the packing solvent at set limiting pressures (Table.1) through a reduced flow tube length in the range of 100mm-500mm, and driving the slurry solvent and packing solvent in the range up to 7000 ml, wherein volume of the slurry reservoir is kept in the range of 10ml-3000 ml, wherein the slurry reservoir is provided with or without flow distributors.
[0027] In an embodiment, the packing solvent from the reservoir is allowed to flow into the column by a predefined limiting pressure of the pumped packing solvent such that slurry solvent is pushed out of the column along with the packing solvent while the packing medium is consolidated into a dense stable bed. In an embodiment, for the packing system, linearity of the limit pressure set in the range 14Psi-10000Psi is ensured such that there is minimal dampening or ebbing effects on the column bed during packing. Further, any line dust is periodically monitored by a grease plate test protocol.
[0028] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawing of the packing system (Fig.1) and a Table 1 on the performance processing conditions and performance results. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.
BRIEF DESCRIPTION OF THE DRAWING
[0029] The detailed description is set forth with reference to the accompanying figure(s). In the figure the use of the same reference numbers in different figures indicates similar or identical items.
[0030] Fig.1 illustrates a schematic diagram of slurry packing system, according to an embodiment herein;
[0031] Fig.2a illustrates a plot representation of column performance parameters, according to an embodiment herein;
[0032] Fig.2b illustrates a plot representation of Van Deemter’s column Height Equivalent to Theoretical Plate, according to an embodiment herein;
[0033] Fig.3 illustrates a graphic representation of performance stability under aggressive pH and elevated temperature conditions, according to an embodiment herein;
[0034] Fig.4a illustrates a graphic representation of back pressure and retentivity at various column temperature in the range of 30ºC-65ºC, according to an embodiment herein;
[0035] Fig.4b illustrates a graphic representation of retention time as a function of back pressure at column 30 ºC, according to an embodiment herein;
[0036] Fig.4c illustrates a graphic representation of retention time as a function of back pressure at column 45ºC, according to an embodiment herein;
[0037] Fig.4d illustrates a graphic representation of retention time as a function of back pressure at column 60 ºC, according to an embodiment herein;
[0038] Fig.5 illustrates a graphic representation of insulin chromatogram using wide pore column packed by the claim system, according to an embodiment herein;
[0039] Fig.6 illustrates a graphic representation of decrease in RT for methyl benzoate, toluene, and naphthalene due to dispersity in pore volume in the range 0.90ml/g-0.97ml/g, according to an embodiment herein; and
[0040] Fig.7 illustrates a graphic representation of typical chromatogram and load ability of semi-preparatory column, according to an embodiment herein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0041] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[0042] As mentioned, there remains a need for developing a method and system for slurry packing of chromatography columns. Referring now to Fig.1, where similar reference characters denote corresponding features consistently throughout the figure, there are shown preferred embodiments.
[0043] According to an embodiment, a method for achieving balanced density slurry-binary solvent is provided. Accordingly, the slurry-binary solvent is prepared by dispersing or suspending chromatographic octadecylsilyl media typified by particle sizes in the range of about 1.5µ-15µ of pore sizes in the range 40A -400A and media having other bonded phases (C-18, C-8, C-4, -CN, -NH2, -C6H5, etc) in about 1g-400g in a binary mixture of solvents selected from a group comprises of tetrabromoethane, CCl4 , CHCl3 or aromatic (BrC6H5) halo hydrocarbon with viscous liquid, wherein the viscous liquid includes glycol, cyclohexanol. According to an embodiment, the volume proportions of halo hydrocarbon carbons and viscous liquid is selected in the range 30% to 70% for packing columns having aspect ratio of 1-120. In a further embodiment, a dispenser which dispenses liquids in predetermined volumes and in short times is used to improve slurry preparation and enhance the production factor. Further, the slurry is ultrasonicated for homogenization and degasification. Thus the slurry is characterized and monitored for suspension time of <60 mins and used during packing.
[0044] According to an embodiment, a packing solvent comprises of low density alcohols and water, wherein the alcohols includes methanol, isopropyl alcohol or combination thereof, wherein the packing solvent is stored in 5-20 liters reservoirs. In an embodiment, the ground water processed and monitored periodically is used for packing the columns, while the commercial graded water is used only for testing the packed columns.
[0045] According to an embodiment, a system for slurry packing of chromatography columns is disclosed herein. Accordingly, the system comprises of an air compressor for supplying air into the system, packing solvent reservoir for storing packing solvent, Haskel pump for air pressure slurry reservoir volume in the range of 10ml to 3000 ml, pressure regulator for controlling the input pressure, manifold pressure snubber for pressure stabilization, pressure gauge capable of measuring up to 20KPsi, a valve for allowing packing solvent through the slurry reservoir into the column, and an adapter outlet having spray distributor that enables uniform flow in the slurry reservoir to which modular columns are attached for packing. According to an embodiment, an end fitting is attached to the column which in turn is attached to the slurry reservoir.
[0046] According to an embodiment, a method of slurry packing columns including controlling air pressure and leak proof in the packing system is provided, wherein the method comprises the step of measuring pressure variance [dp] in the air compressor, and determining characteristic time rated pressure difference [dp/dt] by the line volume fluidically connected to a pump, wherein dp/dt is monitored to keep < 1 and observed as 0.55 for no leak criterion in the present packing system design and also small pressure gradient effects influenced by the compressor pressure cycle is minimally communicated on the pump, to which the column volume is connected to the bed build up while packing.
[0047] In a further embodiment, drawing the packing solvent into the pump at the hydrodynamic volume in the range of 5ml–20ml, keeping the flow rates of packing solvent at a limit pressure rated in the range (as shown in Table 1), keeping the flow tube length in the range of 100mm-500mm, and discharging slurry and packing solvent volume in the range upto7000ml during packing of packing media [slurry], wherein volume of the slurry reservoir is kept in the range of 10ml-3000ml, wherein the slurry reservoir is provided with or without distributors. In an embodiment, the distributors are used for preparative columns to have uniform packing of slurry inside the columns.
[0048] In addition, linearity of the pressure set in the range 14Psi-10000Psi is ensured by controlling the air pressure regulator such that there is minimal dampening or ebbing effects on the column bed during packing. Further, seamless flow tubes is provided for connection between components for a safe and reduced flow path design space, resource feasible, and any tube line dust is periodically monitored by a grease plate test protocol according to which the compressed air from open line is directed towards the layer of grease on a plate support. The dust trapped is monitored and for no dust until the grease layer on a plate is same as the blank.
[0049] According to one embodiment, the pump pressure alone is used to push the slurry into the column and pack the media under pressure in order to achieve a uniform and densely dispersed in columns.
[0050] In an embodiment, as soon as the discharging of slurry solvent and packing solvent reaches within the predetermined range upto 7000ml, subject to the column configuration packed, the packing process is stopped and the column is detached from the flow path. Since these analytical columns can be packed in less than between <10mins to <60mins depending on the aspect ratio, enhanced production capability of the present packing system and the method is of value impact and commercially significant.
[0051] As the packing media determined by the nature of the bonded phase has certain compressibility factor, decompression of the media and the top-off phenomenon of the gel is minimized by processing the column with gel weight in the range of about 1g to about 500g in the slurry for just enough packing under optimal operating conditions of slurry proportion 30%-70% and limiting pressure conditions in the range given in Table.1. Later the end fitting fastened tight at the mouth of the column.
[0052] According to an embodiment, the present invention for packing columns is suitable for analytical columns, wide pore columns mixed bed columns, , semi-preparatory, preparatory and narrow bore columns of various aspect ratios in the range of 1.0–120 which are modular. According to present invention, %RSD of the theoretical plates and asymmetry is low and the method can offer lot-to-lot and batch-to-batch reproducibility in terms of performance as shown in below Table 1.

Table .1 Columns performance
Present invention Columns

Column
Aspect
Ratio Limiting
Pressure,
Psi Flow rate
cm3/min Packing time
mins h
(%RSD) Asymmetry
(%RSD) Void volume (mls)
20-60 1000-10000 1800/min
to
1000/min <10 2.3
(<3) 1.0-1.1
( < 1 )
1.2– 2.85
<25 800-5000 ~30 <2.0
(<2) 0.9-1.2
(<1)
58.0
1-5 800-5000 < 60

[0053] Further, it has been observed that the present slurry packing method and system adopted is effective in producing good and quality analytical columns. In Example 1 columns of aspect ratio 20-60, C18, 5µ, 100? of porous particles, packed using the present system provides void volumes of 1.2mls-2.85mls in close agreement with the prognosticated value of 1.1mls-2.9mls respectively. The void volume for semi-preparative column is 58 close to the expected theoretical value. The void volume is generally calculated as Vvoid = (d2 x p x L x Vpore) / 4000, wherein d is diameter of the particle, L is length of the column, Vpore is Volume of pore, wherein the constant 4000 is an adjustable factor that connotes the ideality with which porous particles are packed in providing structural integrity to the packed bed and inter-particle space for controlled flow volume. The analytical column packed is tested under conditions indicated and the exotic performance parameters such as Void Volume, Back pressure (BP), theoretical plate (N), Tailing Factor (TF), Capacity factor (k’) and Resolution (Rs) obtained is shown in Fig 2a. In the NIST standards test regime of using SRM 870, the present invention column is ascertained as an ideal five finger pattern column case with better ethyl benzene separation coefficient (a=1.6), on Waters 2545 HPLC system.
[0054] Further it has been noticed (as shown in Table 1) that the present packing system and the method offers columns (aspect ratio=20-60) with a mean reduced plate height (h) of 2.3 with a % RSD of <5%. These columns have best asymmetry of 1.0-1.10. In fact, excellent analytical columns presently available have reduced plate height (h) values in the range 2.2 to 2.5 and are related to the methods of packing. Although the columns with 2.0 are exceptional, they are proven difficult or impossible to reproduce due to radial heterogeneity of the packed bed. Van Deemter’s profile (as shown in Fig. 2b) indeed indicates that the present column is endowed with an optimal h = 2.3.
[0055] Further it has been observed that these columns (aspect ratio=20-60) are packed in between less than 10mins or less than 60mins, and the enhanced production capability of the packing system and method is of value impact commercially.
[0056] Further it has been observed that these columns have good pH endurance characteristics in the range 1.2-12 and is shown in Fig.3. The present packing method and system column under acid (pH=2.4) purging condition at an elevated column temperature of 70ºC for 16 hrs shows good acid endurance with a marginal increase of 2% in the void volume and tailing factor by 3% while the theoretical plates decreases by 9%. The present invention column under alkali (pH=10.0) purging condition at an elevated temperature of 40ºC for 16 hrs shows good alkali endurance of N with a marginal decrease of 3% in the first 9.5 hrs and increase by 13% in the remaining time up to 16hrs. The tailing factor decreases by 2.7% while the void volume shows decrease by 24%.
[0057] Further in Example 2, it has been observed that RT shifts with back pressure (BP) and elevated column initial temperature (To). This blue shift of RT is an aberration in accuracies of the methods and or columns employed and are important. The back pressure is found to decrease as the column temperature increased in the range 30C-65C, and is shown in Fig 4a. As does back pressure decrease, the retentivity of a reversed-phase separation of the four reagents also decreases with increase in To, except for uracil which is almost invariant (2.85mmin-2.83min) as a function of To. The chromatograms of a mixture of the said four reagents were recorded at To = 30ºC, 45ºC and 60ºC respectively but different flow rates in the range 0.64mL/min-1.4ml/min. The observed BP corresponding to each of the flow rate were monitored and the variation in the RT each of the component as a function of BP at fixed To are shown in Fig. 4 b, 4c and 4d. The change in Retentivity for a change in back pressure at a constant To is defined as the “Pressure Sensitivity of Retentivity (pRT)” a new column performance parameter hitherto unknown and is expressed as
pRT = (dln RT/dP)To min/psi
Table .2 Pressure Sensitivity of Retentivity of Toluene
To(C ) Flow rate
(mL/min) BP,
(psi) RT,Toluene
(min) pRT = (dln RT /dP)To (min/psi)

30 1.2 2270 11.87
-0.62 x 10-3
1.0 1909 13.94
0.8 1527 18.17
0.6 1143 23.48

45 1.2 1730 10.98
-0.76 x 10-3
1.0 1423 12.02
0.8 1175 16.70
0.6 0859 20.22

60 1.4 1575 06.79
-0.95 x 10-3
1.2 1367 08.45
1.0 1114 10.54

Table 2 indicates that pRT for Toluene decreases with increase in the column To in the range 30ºC-65ºC and has a mean value of pRT = 0.78 x 10-3 min/psi.
Table 3 gives the interpolated retention times for Toluene. The change in the RT with change in the column To at constant BP is defined as the “Temperature Sensitivity of Retention time (sRT )” and is again a new column performance parameter hitherto unknown and is expressed as
sRT = (dln RT/dTo)P , min/ ºC

Table.3 Interpolated Toluene RT and its s
To (C ) BP (psi) RT Toluene
(min) sRT = (dln RT /dTo)P (min/ ºC)
30C
1400
19.80
-2.9 x 10-2
45C 12.40
60C 08.20

[0058] Tables 1 and 2 indicate that the column RT for Toluene is more sensitive to sRT compared to the column pRT. Thus the present method columns have lower pressure and has temperature sensitivities given in Table 1 and 2 and the packing method is also good. In fact, these sensitivity parameters of RT are hitherto unknown and are important new column performance parameters. It is also our aim that attempts are being made to understand the effect of column To on the theoretical plates in terms of HETP.
[0059] Further it has been observed that the columns packed by the present system and method, shows exotic reduced plate height (h), better peak shape, lower pRT and sRT and therefore should provide realistic assays.
[0060] In Example 3, wide pore analytical column of aspect ratio 32.6 of particle size 5µ and pores size of 300?is applied to Insulin (5.8KDa) separation as shown in Fig 5. The present method column separates Insulin and the absorbance band is seen at 17mins lesser than 19.15 mins known to have been achieved by other branded columns.
[0061] Further it has been observed that the packing system and method allows production of columns based on mixed bed strategy of particles with pore volumes of about 1 ml/g advantageous for both SEC and LC like capabilities in a single column.
[0062] Based on the mixed bed technology of packing columns having mixed media particles with distinct pore volumes are packed by using present invention method and system. In Example 4, the mixed bed strategy where several columns of configuration 4.6 x 150 each containing C18 bonded phase of 5µ particle size but media of varied pore volume (0.90ml/g-0.97ml/g) was packed. These mixed bed columns were used to test the separation of the four reagents under identical test conditions of mobile phase, flow rate (1ml/min) and To = 25C. It may be seen from the chromatograms shown in Fig.6 that the variation in the pore volume influences RT. RT values for Toluene decreases from 8.12mins to 6.2mins. The blue shift in the RT values noticed here is purely due to the change in pore volume. Likewise are the variations in RTs for ethyl benzoate and Naphthalene. RT variation is characteristic of the pore volume distribution and hence the associated distribution coefficient Kd=(ti–to)/(te–to) Kd of the constituent reagents. The present mixed-bed columns of the present method and system are advantageous in shorter time analysis worth application in proteomics and and biosimilars and can oovercome column mismatches. The present mixed-bed column is being used to further reduce the RT of insulin (5.8 KDa) far less than 17mins observed with the wide pore analytical column or <19.15 mins observed in other conventional columns and columns as per pharmacopeial analytical methods and assign an ideal Kd.
[0063] Further it has been observed that the Kd specific mixed bed columns produced offers a key to understand specific protein by iterative choice of the mixed bed column for separation methods conducive and separation results intended.
[0064] Further it has been observed that for larger throughput separation of biosimilar or proteins, the method used for packing mixed-bed column is being used in packing mixed bed Semi-Prep columns and preparatory columns
[0065] In Example 5, it has been observed that the present method Preparatory columns of aspect ratio 11.8 have superior performance features with 33% more plate numbers compared to benchmark column with good h, peak shape, higher injection volume, greater load-ability (=1g) per miscibility (as shown in Fig.7, Table 4) with lower resolution distortions (<5%), and are produced in about 30mins by this system and method. The present method semi-prep columns can relatively scale up purification to a sizeable magnitude.
Table .4 Semi-Prep Column Performance

Column
Aspect
ratio Plates
(N) Reduced
Plate (h) Tailing
Factor
(10%) Performance
Remarks

11.8

11500

2.20

1.14
• Higher injection volume
• Superior N @ 10µ, 33% more
• Superior Peak shape
• Greater Load ability: =1g as per miscibility and peak truncation feasibilities.
• Rs Distortion : ~0.5%

[0066] Formation, compaction and conditioning of the Preparatory column of aspect ratio 1-5 containing media (5µ-15µ particle size) is being attempted to pack in less than 60mins under conditions given in Table.1.
[0067] Example A – Analytical columns: About 1g-4g of the C18 or C8 bonded chemistry media of particle size 5µ of pore size 100? or 120? is taken and a slurry solvent of halo hydrocarbon and an alcohol as per present method was added to the media using appropriate dispensers. The 2mins-20mins ultrasonicated mixture through a reservoir is packed into a column of aspect ratio 20-60 modular by using the present packing system, solvents of a mixture of alcohol, water and a pump under conditions indicated in Table 1. After packing, the analytical column is detached from the flow path and subjected to chromatographic test using standard reagent mixture of Uracil, Methylbenzoate,Toluene, Naphthalene under isocratic conditions of mobile phase (Methanol: Water, 70%:30%), flow rate (1mmL/min), ambient temperature.
[0068] Result of Example A - Pressure and Temperature Sensitivity: Example A analytical column of configuration 4.6mm ID x 250mm length, C18, 5µ,100?, packed and having a controlled void volume in excellent agreement with the prognosticated value is used. The effect of column back pressure(BP) and column initial temperature (To) on the retentivity of the reagents like Uracil, methyl benzoate, Toluene and Naphthalene determined using Waters HPLC 2545 system with a oven for elevating the column To up to 150ºC.. The mobile phase (methanol: water 70%:30%) flow rate was regulated at 1mL/min and the BP manifested by the system in the flow path was monitored.
[0069] Example B - Wide Pore Column: About 1g-4g of the C18 or C8 bonded chemistry media of particle size 5µ and pores size of 300? is taken and a slurry solvent of halo hydrocarbon and an alcohol as per the present method was added to the media using appropriate dispensers. The 2mins-20mins ultrasonicated mixture through a reservoir is packed into a column of aspect ratio 32.6 modular by using the packing solvents of a mixture of alcohol, water and a pump under conditions indicated in Table 1. After packing, the wide pore column is detached from the flow path and subjected to chromatographic test using standard reagent mixtures under isocratic conditions of mobile phase (Methanol: water; 70%:30%), flow rate (1mmL/min), ambient temperature and later applied to Insulin separation shown in Fig. 6.
[0070] Example C - Mixed Bed Strategy Columns: About 1g-4g of the C18 bonded chemistry media of particle size 5µ but different pore size such that pore volume of about 1.0ml/g is taken and a slurry solvent of halo hydrocarbon and an alcohol as per present method is added to the media mix using dispensers. The ultrasonicated mixture through a reservoir is packed into a column of desired configuration of aspect ratio 32.6 modular by using the packing solvents of a mixture of alcohol and water and a pump under conditions indicated in Table 1. After packing based on the mixed bed strategy, several columns of configuration 4.6 x 150 each containing C18 bonded phase of 5µ particle size but media of varied pore size (pore volume 0.90ml/g-0.97ml/g) was packed. The mixed bed column is detached from the flow path and subjected to chromatographic test using standard reagent mixtures under isocratic conditions of mobile phase (Methanol: water; 70%:30%), flow rate (1mmL/min), ambient temperature.
[0071] Example D - Preparatory Columns: About 45g-50g of the C18 bonded chemistry media of particle size 10µ is taken and a slurry solvent of halo hydrocarbon and an alcohol as per present method is added to the weighed media. The ultrasonicated mixture through a reservoir is packed into a modular Semi-prep column of aspect ratio 11.8 by using the packing solvents of a mixture of alcohol and water and a pump under conditions indicated in Table 1. It is also observed that the Preparatory columns of aspect ratio 1.0 to 5.0 are also being packed conveniently using the present packing system. After packing, the Semi-prep column is detached from the flow path and subjected to chromatographic test using standard reagent mixtures under isocratic conditions of mobile phase(Methanol: water; 70%:30%), flow rate (1mL/min), ambient temperature. The load-ability of the column was established using methyl benzoate and ethyl benzoate mixture in equi-proportion by mass.
[0072] Accordingly, the present system and method can help in providing high pressure liquid chromatography, fast protein liquid chromatography, and preparatory liquid chromatography columns of aspect ratio1-5 having the benefits of exotic performance characteristics and in volumes exceptional which is of commercial significance.
[0073] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
,CLAIMS:We Claim:

1. A system for slurry packing of chromatography columns, wherein the system comprises of an air compressor for supplying air into the system; at least one packing solvent reservoir for storing packing solvent; a Haskel pump for generating air pressure; a slurry reservoir volume in the range of 10ml to 3000 ml; a manifold pressure snubber for pressure stabilization; pressure gauge capable of measuring upto 20KPsi; a valve for allowing packing solvent through the slurry reservoir into the column; and an adapter outlet having spray distributor that enables uniform flow in the slurry reservoir to which modular columns are attached for packing.
2. A method for slurring packing of chromatography columns of aspect ratio 1-120, wherein the method comprising:
preparing slurry-binary solvent by dispersing and suspending chromatographic octadecylsilyl media typified by particle sizes in the range of about 1.5µ to 15µ of pore sizes in the range 40A to 400A and media having other bonded phases in about 1g to 400g in a binary mixture of solvents selected from a group comprises of tetrabromoethane, CCl4 , CHCl3 or aromatic (BrC6H5) halo hydrocarbon with viscous liquid, wherein the viscous liquid includes glycol, cyclohexanol, wherein the other bonded phase is selected from a group comprises of C-18, C-8, C-4, -CN, -NH2, -C6H5; and
providing a packing solvent comprises of low density alcohols and water, wherein the alcohols includes methanol, isopropyl alcohol or combination thereof, wherein the packing solvent is stored in 5-20 liters reservoirs.
3. The method for slurry packing columns of claim 2, wherein ground water processed and monitored periodically is used for packing said columns.
4. The method for slurry packing columns of claim 2 to 3, further comprising measuring pressure variance [dp] in an air compressor provided in packing system, and determining characteristic time rated pressure difference [dp/dt] by line volume fluidically connected to a pump provided in the packing system, wherein dp/dt is monitored to keep < 1 for no leak criterion.
5. The method for slurry packing columns of claim 2 to 4, further comprising
drawing said packing solvent into the pump at the hydrodynamic volume in the range about 5ml to about 20ml;
keeping the flow rates of said packing solvent at set limiting pressures in the range of 800Psi to 10000Psi through a reduced flow tube length; and
discharging said slurry solvent and said packing solvent in the range up to 7000 ml during packing of slurry in column, wherein volume of slurry reservoir is kept in the range of 10ml to 3000 ml subject to the column configuration being packed.
6. The method for slurry packing columns of claim 2 to 5, further comprising
monitoring tube line dust by a grease plate test protocol, wherein the compressed air from open line is directed towards the layer of grease on a plate support, wherein the dust trapped is monitored and for no dust until the grease layer on a plate is same as the blank.
7. The method for slurry packing columns of claim 2 to 6, further comprises
discharging of said slurry solvent and said packing solvent reaches within the predetermined range upto 7000ml, subject to the column configuration packed; stopping the packing process and detaching the column from the flow path, wherein the column performance are enhanced in comparison with existing method, wherein the packing time is achieved in the range of <10 min to <60min, wherein the reduced plate height (h) is achieved in the range of 2.2 to 2.5, wherein %RSD of asymmetry is achieved in the range of 0.9 to 1.1, wherein void volume (mls) of packing column is achieved in the range of 1.2 to 58.
8. The method for slurry packing columns of claim 2 to 7, wherein the formation, compaction and conditioning of the preparatory column aspect ratio 1-5 containing media particle in the range of 5µ-15µ is packed in less than 60 minutes.
9. The method for slurry packing columns of claim 2 to 8, wherein said method can be used to pack narrow bore column with said bonded phases.