Outstripping all other applications and talking only about healthcare manufacturing, preparative HPLC systems are for a very specific purpose. Their main objective is to isolate, purify, and deliver the target compound like a protein from a solution mixture.
HPLC either called High-Performance Liquid Chromatography or High-Pressure Liquid Chromatography.
Preparative HPLC systems must be more proficient than the analytical HPLC systems to ensure high-purity proteins are fractionated from the column for further processing. They are developed and used for intermediate purification of biosimilars, APIs, and other smaller molecules.
The separation mechanism in reversed phase preparative HPLC systems relies on the interaction between solvent in the mobile phase and stationary hydrophobic resin.
When it comes to HPLC systems, there is usually a lot of confusion regarding the differences in analytical and preparative columns.
We’ll sort-out those first. Later on, we’ll see what are the process steps, different types of elution, and the ways to optimize the efficiency of the columns.
Analytical Vs. Preparative HPLC Systems
|Analytical HPLC||Preparative HPLC|
|Used for analysis of target compounds||Used for preparation by purification of target compound analyzed on analytical HPLC|
|Tells us about the quantity and nature of the component||Separates and collects the component of interest in the required quantity|
|They are Lab-scale||They are Process-scale|
|Have a small column size (column diameter)||Have large column sizes (column diameter)|
|Deals with small particle sizes of stationary resins||Deals with large particle sizes of stationary resins|
|Exerts high back-pressure due to compact size||Back-pressure is somewhat low due to the large scale dimensions of the column|
|Operated at lower flowrates ranging from 0.006 to 0.6 ml/min||Operated at high flowrates range 0.6 to 12 L/min|
|Stand-alone technology||Technology stands on analytical HPLC|
Now that we’ve differentiated both aspects, it is also important to look at different phases within the HPLC system and its working principle.
How Does Column Preparation Work?
Using a moving piston, the column design facilitates the principle of Dynamic Axial Compression (DAC). The use of the piston delivers dynamic compression helpful in column packing, unpacking, and maintaining packing pressure. Though, different suppliers may adopt different operational mechanisms.Column Preparation – Working Principle
For more clarity, let us simply visualize the above statement.
The above image represents the steps involved in column preparation.
- The resin which is a packing material and a stationary phase, first, undergoes slurry preparation in a dedicated and compatible process vessel.
- The slurry then gets transferred to the column by the application of pressure or a suitable pump.
- After the slurry transfer completes, the piston starts to compress the slurry at two different levels.
- Air present in the column space purges out through the piston.
- The solvent in the packing material leaves through the bottom flange and in return packing material experiences compression.
- Column packing is complete and the column is ready to use for commercial manufacturing by maintaining the piston pressure on the packed bed. This is called packing pressure. This helps in avoiding the potential of building vacant spaces inside the bed and this is called Dynamic Axial Compression.
- Once the use of the resin is complete, the column is unpacked by lowering the piston and removing the bottom flange and frit.
Here, important physical parameters of the column design are:
- Column Internal Diameter (mm)
- Stroke of the Piston (mm)
- Maximum Working Pressure in bar(g)
- Maximum Bed Height (mm)
- Bed Volume (L)
How to Specify?
When we see the reversed phase HPLC column, their specifications are generally and combinedly expressed for example as below.
Specification of the Column: C8, 50x1200mm, 7µm, 100Å
The first one, C8 is a chain length of carbon in the stationary phase i.e. resin.
The second one is the column dimension in terms of length and internal diameter.
Third, the particle size of the resin.
Fourth, pore size of the resin.
Particle size for silica-based carbon chain resins generally ranges between 3 to 15µm. Also, there are different carbon chain lengths ranging from C4 to C18.
These resins come with their own mechanical stability dictating the maximum packing pressure they can sustain.
While selecting the resin, it is important to look for its critical aspects and specifications such as:
- Particle Size and Distribution
- Pore Size and Distribution
- Specific Surface Area
- Pore Volume
- Chemical Purity and Stability
- Packing Density
- Coverage etc.
Phases of HPLC System
HPLC systems deal with different chemicals contacting each other at defined parameters. There are two kinds of phases.
- Stationary Phase
- Mobile Phase
Difference Between Normal and Reversed Phase
There are two types of HPLC systems, Normal-Phase (NP-HPLC) and Reversed Phase (RP-HPLC). Let us quickly differentiate them for better clarity. These differences are most commonly understood while this is not an extensive list.
|Normal-Phase (NP-HPLC)||Reversed Phase (RP-HPLC)|
|Superseded by RP-HPLC due to inconsistent reproducibility of retention times||Advancement to NP-HPLC that gives better control over retention time reproducibility|
|Utilizes a polar stationary phase||Utilizes a non-polar stationary phase|
|Utilizes a less or non-polar non-aqueous mobile phase||Utilizes a polar aqueous mobile phase|
|Only organic solvents are used||Solvents like Methanol or Acetonitrile are used|
|The rate of elution is slow in polar molecules and vice-versa||The rate of elution is rapid in polar molecules and vice-versa|
|Analyte elution depends on the increasing polarity of the mobile phase||Analyte elution depends on the decreasing polarity of the mobile phase|
1. Stationary Phase is Polar (Pure Silica)
2. Mobile Phase is Non-Polar (Non-Aqueous Solvents)
1. Stationary Phase is Non-Polar (Advanced Silica with a modified surface)
2. Mobile Phase is Polar (Aqueous Solvents)
Components of Column Module
These RP-HPLC systems have a very complex design on a preparative scale and hence generally classified in different sections.
- Column – Main working frame of HPLC system where separation happens.
- Column Skid – Downstream of the column that handles different components including UV detection lamps, three-way valve assemblies, and related sensors.
- Gradient Pump Skid – These are very heavy-duty pumps required to bring-out the component of interest from the chromatographic bed.
- Remote Control – Basically either HMI or SCADA system to monitor, operate and control the process remotely.
- Hydraulic System – Used to move the jack, up and down during packing and unpacking operations respectively.
These are just the distinctions of critical areas of the HPLC system that again contain additional components explained below.
It is used to close the bottom of the column with a suitable seal and flange arrangement. The frits are used to provide proper liquid distribution over the cross-section of the column through the porous and metallic structures. This in turn helps in achieving consistent and better asymmetry profiles during elution.
Piston is important because it performs three key operations in the column
- Maintaining column’s pressure dynamically while in use
Piston Seal, Compression and Hydraulics
These altogether ensure the complete tightness between the piston and column-wall with the help of a compression system. Apart from the above, the Hydraulic unit performs the following critical functions.
- Controls the piston movement
- Maintains the dynamic compression pressure on the resin bed
There is sequential proportionality between the following.
- Chromatographic Bed Pressure (As applied by Piston)
- Oil Pressure (Associated with the jack)
- Air pressure under dynamic compression
The compression ratio helps to calculate the amount of piston pressure to be applied to maintain dynamic compression during a column operation. In simple form, piston pressure increases linearly with the oil pressure. As a matter of calculation, this ratio should approx. be three (3).
Compression Ratio = (Hydraulic Pressure) / (Piston Pressure) = ~ 3
Based on this, we can say, the hydraulic pressure of 300 bar(g) results in a piston pressure of 100 bar(g).
When the preparative HPLC systems perform neat and clean, pumping systems are the backbones of it. Different applications require different pumping systems.
For example, a pneumatic pump would serve the purpose of dynamic compression on the resin bed and helps in maintaining the jack’s speed movement. An electrical pump would serve the purpose of piston displacement speed during column packing. While the gradient pump would only concerned about handling the flow rate during the process cycle.
Apart from the above critical components, preparative HPLC systems require various supporting components and associated instrumentation.
This includes an oil tank to store hydraulic oil, pressure gauges, safety relief valves, pneumatic distributors, air pressure regulators, pneumatically actuated valves, valve gangs, limit switches, and control panel to operate the equipment with ease.
Slurry Preparation for Packing
The quantity of packing material that should be used in the slurry preparation depends on the following things:
- Bed Height
- Bed Density (Directly proportional to packing pressure)
- Packing Pressure
- Bed Permeability
- Choosing the right solvent for the right phase
- Less viscous slurry
Once these parameters are met, it is then important to note, the use of magnetic stirrers is not encouraged for mixing due to the possibility of particle abrasion. Instead, a recirculation pump loop would help to form a uniform mixture.
For that, packing material should be poured into the solvent instead of solvent in the packing material. Once the slurry is prepared, it is then transferred into the column appropriately.
Buffer Preparation for RP-HPLC
These are the chemical compositions of the solvent that flow through the column bed of resin helping to bind and elute the component of interest at a particular time.
Apart from that, buffers are adjusted to the compatible pH value and added with salts for better column performance.
Generally, there are 2 or 3 different compositions of the aqueous solvent such as Acetonitrile that are used for different physicochemical interactions we’ll see below.
Process Flow: Reversed Phase Preparative HPLC Systems
The process of conducting the column operation for the commercial separation of proteins is pretty straight-forward.
Depending on the nature of the desired component, there exist the following set of operations.
- Equilibration – Performed with less solvent composition buffer to improve the ionic strength of the protein in easily binding the resin.
- Loading – Component of interest preferably in liquid form that binds on the resin.
- Gradient Elution– The main phase that’ll trigger the elution by dynamically increasing the proportion of the solvent composition.
- Buffer Wash – Moderate solvent composition to remove the impurities associated with the product from the resin bed.
- Column Regeneration– A wash that removes any deposits on the surface of the resin including mobile phase contaminants.
The below diagram outlines the overall process scheme of a RP-HPLC system.
This complete process is dependent on the chromatograph running in real-time typically on a SCADA system backed by a UV assembly. We’ll see one-by-one and a typical chromatogram.
1. Column Equilibration – Gives Ionic Strength to Protein
Almost all preparative column resins are equilibrated with a buffer compatible with the compound of interest. These buffers are passed through the resin bed.
The amount of the equilibration buffer to be passed is approx. considered in the range of 5 to 10 times the column bed volume.
Column bed volume can be calculated by using the volume of cylinder formula i.e.
Bed Volume in L = (π * (Bed Height in mm) * (Internal Radius in mm)2 * 10-6) in L
For example, for a bed height of 500mm and column internal radius of 300mm,
Bed Volume = 3.14 * 500 * (300)2 * 10-6 = ~ 141 L
This means the quantity of equilibration buffer should be in the range of 700 to 1400 L for a bed volume of 141 L.
Bed height and resin quantity decided based on the amount of sample that is about to bind the resin. This in turn helps in bed volume calculations.
2. Loading – Binds the Protein
Once the column is equilibrated, the sample load is loaded onto the column bed. As the column is equilibrated, the resin and protein interact, and the protein binds to the resin particles through adsorption.
It is recommended to control optimum flow rate during loading because a higher side flow rate would not allow sufficient time for binding or simply overload the column and a lower side flow rate would unnecessarily increase the time of the loading cycle.
The loading is generally carried out by a dedicated load pump assembled prior to the column. The volume of the load depends on the binding capacity of the resin.
Suppose if the resin is able to bind a maximum of 500gms of the load, then loading a complete load volume that contains 1500gms would be of no sense. Therefore, quantification of the load prior to loading becomes important to overcome the overloading issues.
3. Gradient Phase – Where Elution Occurs
Once the loading step completes, the gradient phase starts. This is the step that separates the compound of interest with high purity. Meaning, most of the weak proteins and impurities are washed off from the resin.
In the gradient phase, the resin is exposed to the increasing proportion of buffer B over a period of time. It is important to note, the gradient is always started with a small % of buffer B preferably less than 10%.
Starting with 0% buffer B may directly impact the chromatographic separation as the binding ability of the protein may deteriorate.
Though the proteins strongly adsorb on the surface of resin particles, they are very prone to leave the surface at higher polar concentrations. The polarity of the aqueous mobile phase depends on the composition of the solvent added into it. As the composition increases, polarity also increases.
The overall operating range for the gradient phase varies from 100% of buffer A to 100% of buffer B dynamically.
To quantify the separation, calculating Retention Time or Rs Value (Resolution) is the simplest method measured as the volume per average width of the respective peak.
At this point in time, it is better to differentiate the different types of elution profiles and their combination.
3.1 Isocratic Elution vs. Gradient Elution
When the composition of the mobile phase remains constant and polar throughout the separation, it is called Isocratic Elution.
While in the Gradient Elution, the composition changes from high polar to low polar. When dealing with protein purifications, gradient gives more clarity in differentiating various peaks without affecting the resolution. If choosing the isocratic elutions, it is important to note; they suffer in detectability in the long run due to band broadening.
This does not mean one should only use either of the ones. The selection of the elution can be determined on the basis of accurate concentrations. The following diagram shows the difference between them.
When we design preparative HPLC systems or simply reversed phase HPLC through the gradient, there are two ways outlined.
3.2 Step Gradient
It is a series of isocratic elutions carried out step-wise at different buffer B compositions. If the concentration at which compound of interest leaves the resin is known, a step-by-step isocratic elution approach saves time in future separations than following gradient elution on a regular basis.
It is considered ideal during process scale applications for which desired resolution can be derived because this method utilizes simple instrumentation clusters effectively. Also, when the separations are carried out in the lower regime of resolutions step gradients are generally preferred.
3.3 Continuous Linear Gradient
When dealing with a larger regime of resolutions, the continuous gradient is the choice of separation. In this type, the composition of the mobile phase changes linearly with time.
Unlike isocratic elution, this gradient keeps band broadening to a minimum and hence helps in higher detectability.
4. Buffer Wash – Separates Other Associated Impurities
A buffer wash of 100% composition should flow through the column bed upon completing the gradient phase.
This helps in removing any protein traces that may have stuck on the resin and also the associated impurities.
Generally, the sudden change in mobile phase composition may damage the packing quality and hence this wash helps in avoiding that. Also, this step helps the column to set it up for further use.
5. Regeneration – Helps in Removing Deposits
After washing, regenerating the resin bed for the next run is critical and requires a higher composition of the aqueous polar solvent suitably a separate buffer C.
If this step is not performed, the issues would be catastrophic in terms of column performance including;
- A sudden increase in back-pressure
- Changes in retention times
- Poor separation
- Poor peak broadening
These signs indicate that the resin has been impacted by the compound deposits or precipitations on its surface. Flow should be on the lower side and up to 10 column volumes.
A proper regeneration technique should be applied either in the normal flow or reversed flow without affecting the column performance.
Remember, Backpressure should not be more than packing pressure. This ensures the column is not clogged and can be used for separation purposes. If backpressure > packing pressure, the resin should be washed with acid in an upward direction ensuring complete residue desorption from the resin surface. Additionally, filters to be replaced if chocked both for load sample and mobile phases.
Reversed phase i.e. stationary phase resins, should be stored in a mixture of water and solvent. Care should be taken to avoid using any additives that may precipitate when stored.
When the operation of the column is complete, it is generally washed for 10 to 15 minutes in proportional composition.
Now that you’ve seen the process steps for reversed phase preparative HPLC systems, let us see an example of a typical chromatogram.
The chromatogram runs in real-time during the complete operation of the process. At this point, we must understand few indicators that are important to understand how the column performance is dependent on various factors.
Let us start with the critical process parameters associated with preparative HPLC systems.
Critical Process Parameters
When it comes to protein separation by reversed phase chromatography, it is important to understand that small fluctuation in any of the process parameters has a critical impact on product quality.
It may ultimately denature the protein of interest due to its sensitive nature. Following are the commonly considered critical process parameters for preparative HPLC systems.
- Mobile Phase: Choosing correct organic modifier such as Acetonitrile which are UV transparent
- Temperature: Increase in temperature will cause a decrease in viscosity of the mobile phase. This affects the compression ratio also due to changes in oil properties.
- Flow rate: Affects dynamic binding capacity (resin binding kinetics) especially during loading of process scale preparative HPLC systems.
- Gradient-Elution profile: Typically linear gradient method is preferred for preparative HPLC systems. Meaning, the gradient flow will be from higher to lower polarity levels.
- Stationary and Mobile phase physicochemical interactions
Evaluating Column Efficiency (Half-Height Method)
One concept while evaluating the success or efficiency of the column is called Height Equivalent to Theoretical Plate (HETP). This is expressed in terms of the number of theoretical plates (N).
In measuring the efficiency of preparative HPLC systems, this dimensionless number tells us about the column packing attributes such as;
- Column packing is good or needs repacking
- Whether the packing material is intact
When dealing with protein manufacturing such as Insulin, the peak of the protein in the chromatogram follows the Gaussian path. In such cases, a valid and most preferred way to estimate the column performance is by using the peak width at 50% peak height also called Half-Height Method.
Let us plot a graph considering the time of injection at around 48 minutes. Just to show differently than our previous chromatogram where the time of injection was 20 minutes.
Column Efficiency for preparative HPLC systems is calculated in terms of number of theoretical plates (N) per column as below.
N = 5.54 * (tR / W0.5)2
Once the number of theoretical plates per column calculated, you may also calculate H i.e. Height Equivalent to Theoretical Plate (HETP) which is the number of theoretical plates per meter of given column length.
H = L / N
tR – Retention time
H – Height of the curve
W0.5 – Width of the peak at half-height
W – Width of the peak 10% above the baseline (just at the peak tailing)
L – Column Length
N – Number of theoretical plates per column
Though we’ve discussed the half-height method for evaluating column efficiency, there are other alternative methods available to derive the same such as:
- Tangent-Line Method → N = 16 * (tR / W)2
- Modified Gaussian Method → N = 41.7 * ((tR / W0.1)2/((b/a)+1.25)) where W0.1 – Width at 10% height.
- Area Height Method → N = 2π * (tR.H / A)2
Using any of the above methods bound to give slight variations in “N” depending upon the peak characteristics and shape.
However, the half-height method is often adopted for automated software such as SCADA that evaluates column efficiency seamlessly.
Peak sharpness increases with the increasing number of theoretical plates (N) per column. This means, more the number of theoretical plates, the more is column efficiency.
1. How to Optimize the Column Efficiency?
→ Flow rate or solvent velocity calculated at reduced plate height
→ Particle Size of the resin
→ Length of the column (L)
2. What is the Desired Elution Pressure? How to Tackle Back Pressure Issues?
The best way to confirm that no overpressure issue with the column exists is through the theoretical estimation of the elution pressure. Certain pressure drop (ΔP) will surely be there in the column and it can be estimated as below.
ΔP = ((k0*u*L*η) / (dp2))*10-6
dp → Particle diameter
u → Flow Velocity
L → Column Length
η → mobile phase viscosity
k0 → permeability constant generally considered as 600 for spherical beads
This equation is derived from Darcy’s law of pressure drop. If the pressure drop is significant, it might be the situation of deteriorated packing material. Though in such cases, expert opinions are advised.
Peak Asymmetry Factor (As)
This is also one of the important factors in measuring column efficiency that tells us the behavior of the peak symmetry in terms of an offset.
This is expressed as As and can be calculated by dividing the centre of the peak into two parts equally at peak width (W). Suppose the left part from the division is “a” and the right part is “b”. Then Peak Asymmetry Factor can be calculated as;
As = b/a
When this ratio turns out to be 1, the peak is called perfectly symmetrical. If <1, termed as fronting peak and if >1, termed as tailing peak.
Evaluating Packing Efficiency
A poorly packed column can significantly drop the performance of reversed phase preparative HPLC systems. This is one way to evaluate whether the column has been packed well.
A Dutch physicist and engineer Van Jozef van Deemter related the HETP of the column to the linear velocity of the mobile phase through mass transfer, chemical engineering thermodynamics, and reaction engineering.
The equation he provided is called Van Deemter’s Equation and expressed as;
HETP = A + (B/u) + (u*C)
A – Eddy diffusion (which results in peak broadening)
B – Longitudinal diffusion coefficient
C – mass transfer between mobile and stationary phase
u – flow velocity
The last term in the equation when multiplied with Peclet Number is called Rodrigues equation. This is an extended version of Van Deemter’s equation that indicates the efficiency of the resin bed for large pore sized permeable particles.
Just to give you an idea and talking in terms of reversed phase preparative HPLC systems,
A < 1 → Well packed column
1 < A < 3 → Column packing can be accepted conditionally
A > 3 → Bad quality of either the packing material or packing itself
Similarly, C > 0.3 → Inadequate mass transfer between mobile and stationary phase.These values should not be considered exactly same and may vary depending upon different applications.
Relation Between Linear Velocity and Flow Rate
In preparative HPLC systems or any chromatographic system, standardizing the flow rate for different columns is expressed by using linear velocity (m/s or any suitable unit) as a multiplication factor. Linear velocity is the speed at which the mobile phase passes through the column.
During process scale-ups or scale-downs, considering the same flow rate that was used in the previous column of internal diameter different than a new one is not a practical solution. So to compare the equivalence between the two columns, linear velocity generally kept constant.
Instead of using the same flow rates, the linear velocity is kept constant to adjust the flow rate for the new column dimensions proportionally. The following formula is used to calculate the new flow rate expressed as the square of the ratio of the column IDs;
New Flow Rate (LPM) = Previous Flow Rate (LPM) * (New ID / Old ID)2
- Suppose, the flow rate for the previous column having an internal diameter of 800 mm was 15 LPM. Now the changed column diameter is 1200 mm. According to the above formula
New Flow Rate (LPM) = 15 * (1200/800)2 = 15 * 2.25 = 33.75 LPM.
- Suppose, the flow rate for the previous column having an internal diameter of 1200 mm was 33.75 LPM. Now the changed column diameter is 800 mm. According to the above formula
New Flow Rate (LPM) = 33.75 * (800/1200)2 = 33.75 * 0.45 = 15 LPM.
Retention or Capacity Factor (Kc)
When dealing with protein purification, it is important to keep a record of retention factors of the different compounds including the one of interest.
If they are the same, impurities will also follow the compound of interest and would yield impure protein at the time of elution.
Also called the Capacity factor, it is the measure of proportional time a load sample stays in the stationary phase than the mobile phase. It is different than the binding capacity.
The mass of the solute in a specified amount of stationary phase that is able to bind at specified conditions is called Binding Capacity. The measure of proportional time a solute spends in the stationary phase than the mobile phase is called Capacity Factor (Kc).How Binding Capacity is Different Than Capacity Factor
For every peak that logs into the chromatogram, retention factor or capacity factor for a loading sample can be estimated by:
Kc = (Number of moles present in stationary phase / Number of moles present in mobile phase)
It is the ratio of the retention factors expressed at particular mobile phase composition as;
α = kb / ka = Vb /Va
kb – retention factor of a compound that retains on the resin for a long time
ka – retention factor of a compound that easily leaves the resin
Vb – retention volume of a compound that retains on the resin for a long time
Va – retention volume of a compound that easily leaves the resin
It is equivalent to the relative retention of the solute peaks. In other words, selectivity is the ability of a reversed phase preparative HPLC system to separate two analytes from each other.
This is the function of selectivity, retention factor, and the number of theoretical plates (N). Resolution Rs defines the separation of chromatogram peaks which is the main purpose of chromatography.
Changes in Resolution could occur due to properties of the resin when equilibrated, washed, flow rate, load volume, and buffer composition.
Distance between the centers of the two elution peaks in terms of Retention time per average peak width. This is measured by considering the contribution of selectivity, efficiency, and retention factor
The formula to calculate Resolution is as below.
α – Selectivity (defined earlier)
k – retention factor
N – number of theoretical plates
Yield is defined as the quantity of protein recovered with respect to the quantity of protein loaded. It is used to evaluate the productivity of preparative HPLC systems.
Yield (%) = (Quantity of Protein Recovered for Next Stage / Crude Protein Loaded)*100
Common Problems and Troubleshooting
Poor Plate Counts
Chromatograms results in poor plate counts due to overloading of the injection volume onto the column resin. General practice is that the injection volume should not be more than 10 to 15% of the loading flow rate.
This means sample quantities of 0.01 to 0.015 mg should be loaded per ml of column volume at max.
Another reason for poor plate count is dead space. Dead space is the overall volume occupied by injector assembly, tubing, UV detector assembly, and other mechanical components such as connectors, filters, etc. Hence proper assembly of all the components is the key.
Ghosting or Ghost Peaks
Have you seen unknown peaks that neither resembles your compound of interest nor associated impurities? If so, please be assured that your mobile phase has a poor quality.
The organic impurities in the mobile phase may bind to the resin at significant levels such that during buffer wash after elution, these contaminants desorb from the surface of the resin and make an appearance in your chromatogram known as Ghost Peak.
However, they do not cause any harm in the short term but can severely damage the resin in long term. Just use a high-quality mobile phase and monitor regularly for such peaks.
It is the primary sign of partially chocked frits. This is caused when the loading sample and mobile phases are injected without using a filter that may contain certain physical components irrelevant to the desired separation.
Using proper filters preferably of small pore sizes would prevent the physical impurities from being injected onto the preparative HPLC systems.
Specifically, at the top of the bed, the chances of precipitation of compound of interest are high. In this scenario, it is important to clean the filters and the column. See FAQ-2 above for more details.
As discussed earlier, poor resolution can be a result of poor selectivity. Sometimes, the reason might be poor column packing and column overloading.
Evaluating column efficiency and repacking the column if required, are the choices to overcome this problem. At the same time, cleaning and performing the regeneration step should be helpful.
When the elution strength of the injecting solvent is more than the mobile phase, peaks can split or break because the two compounds may be separating at almost the same time.
To avoid this scenario, using a smaller resin particle size and greater column length will produce better separations.
Spikes In Chromatogram
Spikes coming in the chromatogram show UV baseline rises from the initial value to the next value irrespective of changes in the concentration of the phase.
The main cause of this issue is related to the air bubbles trapped in the UV cell. Degassing the UV assembly is the choice of treatment in such cases.
Periodic Column Cleaning
When it comes to column fouling contaminants, periodic cleaning is recommended. Remember the signs mentioned in the regeneration phase earlier?
When those are increased to a significant level, a cleaning with low pH mobile phases are preferred to wash off any contaminant on the reversed phase silica-based resins.
These types of resins contain Silanol groups that form gel aging when exposed for longer durations to aqueous mobile phases.
General way of column cleaning is as below.
- Equilibrate the column by passing multiple column volumes of buffer A at a low flow rate with a minute proportion of any suitable acid in it. Sometimes, alkalies can also be considered.
- Run a dummy gradient starting from 100% buffer A and ending on 100% buffer B again containing a minute proportion of suitable acid. Remember to execute on approx. 25 column volumes.
- Perform column equilibration similar to the above step but this time starting from 100% buffer B and ending on 100% buffer A.
- Finally, re-equilibrate the column using buffer A with multiple column volumes around 10x.
Custom made automated software preferably PLC and SCADA combinations would help in delivering consistent and quality performance.
The monitoring software should possess the following features and compliances at-least to consider the process as reliable.
- Robust Control on Critical Process Parameters
- Column efficiency measurement tools
- Dynamic chromatograms with required filters
- Column Pressure and Temperature Indicators
- Automated Backflush Sequence Provision
- Alarms and Interlocks as per Process Flow
- Facilitating smooth fraction collection during elution
- Flow Totalizers for easily knowing how much column volumes we’re processing
- Hierarchy-based User Access Level Rights
- Qualified the Computer System Validation (CSV) prior to commercial use
- Compliant with 21 CFR Part 11 and GAMP5 Guidelines
While the list is non-exhaustive, using these automated features in your reversed phase preparative HPLC system can really give a boost in terms of performance optimizations and regulatory compliance.
This definitive guide for RP-HPLC dealt with almost every aspect including column design, proper development, process application, and optimizing your preparative HPLC systems. Right from fundamentals to the troubleshooting.
When these systems are used in the downstream manufacturing of APIs, the collected fractions are further processed for polishing the proteins till they get lyophilized.
Please note, the concepts explained above are as per the past professional experience with various organizations that were practicing preparative HPLC systems. You may follow a different path while dealing with other products depending on your needs. But the fundamentals mentioned above remain the same.
It would be interesting to know how you resonate with this article. Do you feel something important is missing? How do you conduct your preparative HPLC systems? What problems do you face? Comment below.