Photometers & Turbidimeters

In the biopharma sector as well as in other industries that rely on accurate process measurements, the continuous calibration of online UV analyzers is an established practice. But a deeper understanding of light absorbance and advances in technology prompt a pertinent question: Is there a better, more efficient way?

Calibration vs. Validation: Reviewing the Facts

Understanding Light Absorbance

The science of light absorbance is pretty straightforward. For instance, when 90% of light from a monochromatic source is lost after passing through a substance—meaning only 10% makes it to the other side—that substance exhibits an absorbance of one. Measurement of absorbance is achieved with instruments incorporating a photodiode, a device that reacts proportionally to the amount of light hitting its photoactive area. Consequently, a one absorbance change in received light equates to a log decade change in output from the diode—assuming, of course, that there’s a high-quality electronic circuit around the photodetector.

Continuous Calibration Drives Up Cost and Consumes Time

UV absorbance analyzers are widely used in biopharma separation processes to detect protein product presence in eluents from chromatography columns and in permeates in filtration systems. These devices are crucial in maximizing product yield/quality and maintaining regulatory compliance.

However, the continuous need for calibration is both costly and time-consuming. If a photodiode’s output truly reflects the absorbance of monochromatic light, an inline absorbance instrument, or photometer, should natively read out an absorbance that doesn’t require calibration. So, what prompts the need for this constant calibration?

Unpacking the Need for Calibration

The root of the calibration conundrum can be traced back to a few factors, with the quality of the optical bench being a major one. For accurate absorbance measurement, monochromatic light is essential. Many inline UV absorbance photometers employ light sources—like mercury vapor lamps—that do not generate monochromatic light. These lamps emit a plethora of light at a multitude of wavelengths. Curiously, they don’t generate light at 280 nm, the most popular wavelength utilized in biopharma separation work.

Filtering Brings Series of Drawbacks

Therefore, aggressive filtering is necessary to create a pseudo monochromatic peak for measurement. Filters manufactured using multi-cavity coatings on glass are most often used to reduce wavelengths outside of the wavelength of interest reaching the photodetector, but this does come with trade-offs. The more aggressive the filtering, the tighter the peak bandwidth butthe lower the energy of the passed light. Any remaining stray light passing through the filter can quickly affect the measurement, leading to non-linearity even at levels nearing one absorbance.

The LED-Based Photometer Solution

The solution to this problem lies in LED-based photometers, like the Kemtrak DCP007-UV photometer. These devices generate light with a much, much lower incidence of stray light, making it much. much easier to create a pseudo monochromatic light peak. With less stray light, absorbance can be measured more deeply, with greater linearity and the signal becomes a much better representation of absorbance.

Instruments that exhibit this linear response to absorbance remove the need for calibration and instead require only validation—a periodic check to confirm that the instrument is operating as expected. Successful validation is easily achieved by simulating absorbance in the instrument’s optical path. A change in measurement that falls within an expected response verifies that the instrument is functioning as expected.

Such a move away from calibration to validation is not possible with mercury vapor lamp-based instruments due to the need for calibration factor changes necessary to mitigate the effects of stray light. However, It is achievable with LED-based instruments as they intrinsically read in absorbance.

The Validation Advantage

Validation brings a range of advantages, including:

• Increased Accuracy: Validation ensures that the instrument operates as expected, reducing the chances of errors introduced during calibration by different technicians.
• Greater Consistency: By simulating absorbance in the instrument’s optical path, validation provides a more consistent method of ensuring that the instrument is working properly.
• Lower Costs: The process of calibration can be expensive and time-consuming. Validation, on the other hand, typically requires less effort and resources, leading to cost savings. As a side note, take the example of a large pharmaceutical company in Scandinavia that recently addressed the cost of calibrating absorbance monitors by implementing LED-based units, rather than mercury vapor lamp-based instruments, as it expanded its production capacity. The savings that the company will realize for the 60+ units in question will be substantial, even before taking into account potential product yield improvement and reduced loss advantages.
• Enhanced Linearity and Depth of Absorbance Measurement: LED-based photometers measure absorbance more deeply and with greater linearity than traditional lamp-based units. This enhanced performance in absorbance reading also means that routine calibration is no longer necessary, with validation of the instrument’s performance being the only requirement.
• Simplified Process: Validation is a simpler process that only requires simulating absorbance in the instrument’s optical path and observing whether the instrument responds as expected. This straightforward method is less susceptible to errors or variations in technique.
• Streamlined Compliance: Calibration often comes with a host of regulatory requirements that can be difficult and costly to maintain. Validation, however, can often streamline compliance processes, making it easier to meet industry standards and regulations.

Reading tip: For a comprehensive understanding of the impact of photometer design on photometer validation methods, read this white paper.

It’s Time to Future-Proof Your Business

As the landscape of the biopharma sector and other industries requiring accurate measurements continues to evolve, it’s critical to stay abreast of advancements that enhance efficiency and accuracy. Adopting LED-based photometers is not just a viable alternative to traditional methods—it’s a forward-thinking solution that redefines standards, reduces costs, and drives process optimization.

We encourage all decision-makers, process engineers, and quality control specialists to consider this shift from calibration to validation. It’s a step towards future-proofing your processes and keeping up with the pace of innovation.

Contact South Fork

Interested in learning more about how LED-based photometers can revolutionize your operations? Feel free to reach out to us at South Fork Instruments. Our team of experts is ready to provide insights, answer questions, and guide you through this transformative journey. Contact us today to learn more by filling out this form or calling us at (925)461-5059.

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Having the right analyzer can make a big difference to separation performance

Reliable and repeatable post column UV measurements are a minimum requirement for column chromatographic separation processes to ensure good protein fraction purity and maximum yields. With the lack of any cost-effective, industrialized inline spectrophotometer scanning systems for UV measurement, industry has tended to go for discrete wavelength analyzers/photometers to perform this vital task. The question is which analyzer is best equipped to provide real-time single or dual wavelength UV analysis for instant visibility and improved control of the separation process?

Column chromatography is widely used in bioprocessing to separate protein molecules from concentrated process fluids. The center of such a chromatography system is a column, filled with a media specific to the separation being carried out. There are a variety of methods employed in chromatographic separation — gel filtration, ion exchange, hydrophobic interaction, affinity to name a few — but all have the same need to accurately and distinctly identify UV absorbance peaks post-column.

The Role of the UV Absorbance Analyzer

Separation through chromatography requires highly specialized equipment to ensure maximum yield and purity, and a typical system will include a variety of instruments and sensors. A UV absorbance analyzer at the column outlet detects protein coming off the column (because nearly all proteins absorb UV energy at or around 280nm). The analyzer measurement cell is fitted in the outlet line of the column, and as proteins exit the column, the absorbance output “peaks” up, allowing protein-rich solution to be diverted for collection.

Zero Hold-Up Volume for Complete Clarity

One important factor often overlooked is that a UV analyzer should have zero hold-up volume to ensure crisp, sharp peaks are detected. UV analyzers utilizing measurement cells with internal hold-up volumes can lower the purity of the collected protein because of dilution. Dilution blurs sharp peak detection lines, making collection trigger points far more difficult to define.

Dilution effect on a protein “slug” passing through in an optical cell with hold-up volume

Effect on UV absorbance peak sharpness

Measuring Deep Absorbance

Advances and improvements in bioprocessing methods have led to protein products being expressed and concentrated to higher and higher levels. Higher protein concentrations mean higher inline absorbance measurements. Therefore, UV analyzers need the ability to measure deep absorbance.

While absorbance measurement following Beer-Lambert Law is essentially linear (up to a certain point, anyway!) as long as the UV-light source being used is monochromatic, some inline UV analyzers use light sources that generate light at wavelengths other than 280nm; this is particularly true of analyzers/photometers that use mercury vapor arc lamps. Light energy from these additional wavelengths enters the measurement path as stray light to create artificially low absorbance readings and generates a non-linear result (compared to offline sample analysis) as concentration increases. At higher concentrations, the analyzer response becomes increasingly non-linear, eventually flat, as the stray light becomes the dominant part of the measurement.

Plus and minus

As a result, optical filters are used to block stray-light energy in many instruments, but this has both positives and negatives. On the plus side, analyzer linearity is improved because the light energy passing through the filters focuses the absorbance measurement more upon the wavelength of interest.

On the minus side, band pass filters also block a significant part of the light energy at the wavelength of interest, so light sources need to be more powerful. The design of many inline photometers has the UV light source installed directly on the measurement cell. High levels of UV energy (from all wavelengths including 254nm) and the associated heat generated by the lamp are introduced to the flowing stream when the analyzer is operating, making them less than ideal, particularly for fragile protein molecule work.

Performance, Traceability and Validation

Baseline drift, due to the decay of the mercury vapor lamp is a common feature of inline UV absorbance analyzers. In addition, mercury light sources are inherently “noisy” as the internal arc generated by high voltage naturally flickers and moves. These two features mean that baseline adjustment or zeroing is continually needed and data recorded from the analyzer will often exhibit spikes along trend lines on charts.

Because of these factors, it is necessary to constantly “calibrate” and adjust these units. The best way is always to use a liquid standard, but some liquid standards (Potassium Dichromate, for instance) may not be chemically compatible with the process and process samples of varying absorbance may not be available. A suitable substitute is to use traceable optical filters certified by a third-party laboratory. Laboratory benchtop spectrophotometers are calibrated/validated using such filters and the same ones should be used for the online analyzer to provide direct linkage between online and offline sample testing results. Custom fit filter devices for validation purposes are problematic as this linkage cannot be properly established.

So Which Analyzer Comes Out on Top?

Everyone agrees that accurate, reliable, and repeatable post column UV measurements are a minimum requirement for chromatographic separation to ensure good protein fraction purity and maximum yields. To achieve these goals, the desirable features of an inline UV analyzer seem to be:

• Zero holdup volume
• Deep absorbance measurement capability
• No zero/baseline drift
• Has a linear measurement performance according to Beer-Lambert Law
• Low energy light source
• Low heat light source
• Ability to validate to the same standard as a laboratory/benchtop spectrophotometer

A closer look at Kemtrak DCP007

Taking the above into account, the unit that immediately stands out in the crowd is the Kemtrak DCP007 analyzer. This particular unit utilizes:

• Zero holdup volume inline measurement cells that ensure crisp peak detection without dilution effects.
• Solid-state light sources (LED’s) to remove the problem of stray light blockage and allow for measurement depth up to 4.5A at 280nm (@1cm optical path). With a short pathlength, it’s possible to reliably measure up to 90 OD without “peak clipping”.
• A unique optical bench design that monitors both emitted and received light when in operation. Emitted UV light energy is maintained at a continuous level with the result that zero/baseline drift is eliminated.
• True absorbance (A) as its baseline measurement. Measurement linearity follows Beer-Lambert Law until the process concentration itself introduces non-linearity from molecule density.
• LED light sources that emit “cold” light. Because emitted light is monochromatic, heavy optical filtering is not necessary. Accordingly, light loss from optical components is very low and therefore, emitted energy is low. Because of its low heat and extremely compact nature, the light bench is installed in the analyzer electronics enclosure and connected to a very compact measurement cell using fiber optic cables. Additional benefit of the low energy light output is an exceptionally long light source lifetime.
• The ability to be validated to the same standard as a laboratory/benchtop spectrophotometer. Using cuvette style traceable filters, the baseline measurement behavior of the analyzer can be tested and observed, providing absolute confidence in measurement.

The design of photometer instruments has a significant impact on their applicability. Recent design improvements provide better overall measurement performance and long-term measurement stability. So, when evaluating photometers, it helps to understand how certain features affect the validation and verification methods.

In our new tech brief, Validation of Inline Photometers for Increased Measurement Confidence, we explore the designs more in-depth as well as discuss their pros and cons. For a quick introduction to a few aspects of photometer instrument design, keep reading this post.

Simply put, an industrial photometer is used to measure light after it has passed through an industrial process stream. Photometers can be configured for a variety of purposes, with the configuration and technique used depending upon the specific measurements needed. This makes them applicable to a wide range of applications in process analytical chemistry.

Understanding the impact of Beer’s law

The science at the core of photometer instrument design is Beer’s law. Also known as the Beer-Lambert law, it relates the attenuation of light to the properties of the material through which light is traveling. Since different chemicals absorb light at different wavelengths, the application of Beer’s law helps determine the accurate concentration of a chemical solution.

For the majority of applications where absorbance characteristics obey Beer’s law, a simple zero-plus-one sample point will be sufficient to calibrate an online photometer. The key to obtaining a signal that is proportional to concentration is that the light reaching the photodetector must be monochromatic (single wavelength).

But there are exceptions that require non-linear calibration: Beer’s law does not describe the behavior of concentrated solutions (greater than 10-2 M) due to interactions between the absorbing molecules. It also does not apply when measurement is made at a wavelength where the slope of the sample absorption curve is very steep. In such cases, a non-linear calibration should be used. Industrial photometers typically include non-linear and piecewise linear calibration models.

Considering a few photometer instrument designs

Let’s take a look at a few design variations:

Spectrophotometers

Spectrophotometers produce monochromatic light by using either a single photodetector with mechanically moving grating or prism, or a diode array detector if the instrument is more modern. They use almost exclusively broadband light sources such as halogen for visible (VIS, 400-700nm) and near-infrared (NIR, 700-2000nm) and deuterium for ultra violet (UV, 200-400nm).

Traditional process filter photometers

Traditional process filter photometers use narrow bandpass optical filters in conjunction with broad spectrum, high energy incandescent, halogen (VIS, NIR) and mercury vapor (UV) light sources to generate a facsimile of monochromatic light. In traditional designs, the light source is hot and a filter in close proximity to it is subject to potential damage over a relatively short period of time.

Modern filter photometers

Modern filter photometer designs utilize high performance specific peak wavelength light emitting diodes (LEDs) instead of broadband light sources and narrow band filters to produce tight, narrow band light emission. Unlike traditional process filter photometers, the LED source is cold and and does not damage or degrade other sensitive optical components in close proximity. A further advantage is that the process itself is no longer exposed to high energy light that can be potentially damaging to the process itself. This is particularly true for applications such as protein detection in bioprocessing that utilize UV light for the measurement

Addressing the issue of stray light

A problem with process filter photometers is the issue of stray light. It is the effects of stray light that often lead to dissatisfaction with photometer performance when deployed into a process environment. Typical bandpass filters only block to approximately 3 AU (0.1% of the total transmitted light) outside of the pass band and all remaining light below this level passes freely through the optical filter, creating non-linearity in absorbance measurement and limiting instrument range.

Narrow bandpass filters

Narrow bandpass filters are constructed to allow light to pass through a very small region or slot, typically 10nm wide, around a specific peak wavelength (the wavelength of interest), while blocking light from passing at all other wavelengths. When narrow bandpass filters are used in combination with process filter photometers that utilize LED light sources, there is virtually no stray light outside of the pass band region around the wavelength of interest.

As a result, LED light source photometers adhere to Beer’s law and provide native units of absorption as their baseline measurement without the need for additional absorption calibration. This means that any engineering unit can be correlated to absorbance while leaving a possibility for confident validation of the instrument’s performance.

Watch for decay

But a word of caution is warranted. Bandpass filters, while generally stable, can exhibit signs of decay over time with changes in peak transmission and stray light blocking capability caused by environmental factors e.g., moisture or erosion from the light source itself. This is particularly true in traditional design filter photometers. Since bandpass filters essentially define the operating wavelength and performance of a process photometer, it is important to ensure they meet or exceed the original specification from the manufacturer.

Impact of broad spectrum light sources

As mentioned above, when the light reaching the detector is not monochromatic, Beer’s law does not apply. Photometers that use broad spectrum light sources necessarily produce a significant amount of stray light passing beneath the out-of-band blocking capability of the filter and onto the detector. Such photometers must, consequently, be calibrated at multiple points for the instrument to work in units of absorption, noting that absorption is proportional to concentration. This requirement for signal correction presents some specific challenges when validating instrument performance.

Summing up

In sum, an understanding of the stray light issues surrounding filter-based photometers can greatly improve the approach to field validation and can enhance measurement accuracy. Using multiple validation points can help describe non-linearity exhibited by a photometer and allow action to be taken to correct it. Provided stray light impact is understood, highly reliable validation measurements can be obtained with a resulting high confidence in the measurement results of the instrument itself.

Do you have questions for us about photometers, or other measurement instrumentation and analyzer solutions? Please contact us. Our experts stand ready to assist.

Few factors are more important to operators than ensuring an instrument is working to specification and providing the correct measurement to an overall control scheme. 

Finding the right method for process photometer validation is therefore a priority. Operators can realize significant gains by achieving that aim, including ease of equipment maintenance, replication of maintenance efforts, and increased confidence in the instrument’s measurement performance. 

The most typical validation method that operators now use is verification of instantaneous reported instrument reading against a process sample measured offline. But the method comes with certain drawbacks:

• It is limited to only one sample concentration at a time 
• It may not provide a true indication of how the instrument will perform under changing process conditions. 

To more closely examine the pros and cons of various photometer validation and verification methods, we created a tech brief for download, “Validation of Inline Photometers for Increased Confidence”. It provides an in-depth look at key variables, including:

• Photometer Method & Design 
• Validation
• Validation of Wavelength and Blocking
• Validation of Photometric (Absorbance) Performance
• Optical Pathlength and Error Assessment

In this post, we will feature a sample of the various methods, but before we begin, let’s look at the definitions:

What is a photometer?

A photometer is an optical instrument designed to measure the absorption of light at a given wavelength after it passes through a fixed distance of sample. Different chemicals absorb light at different wavelengths, and through the application of Beer’s law, the concentration of a chemical in solution can be accurately determined. Photometers are highly applicable to a wide range of applications in process analytical chemistry.

What are different types of photometers?

Filter photometers

Filter photometers are instruments designed to operate at one or more fixed wavelengths e.g. 280, 300, 330, 400nm. 

• They are used as an online, real-time process instrument. 
• They are a simpler and therefore more cost-efficient instrument configured to continuously monitor a fixed process stream as part of an overall plant control system.
• The expectation is that they will operate unattended over long periods of time without operator intervention or the need for maintenance and servicing. 
• Process photometers use specialized industrial in-line measurement cells or immersion probes which are designed to withstand harsh process conditions e.g., extreme pressures and temperatures, hazardous/explosive and radioactive environments.

Spectrophotometers

Spectrophotometers allow monitoring of a range of wavelengths within instrument specifications, e.g. 400-800nm. As they provide flexibility when working with a wide range of different samples, they are commonly used in chemical laboratories.

• They are precision instruments, designed to be manually configured and operated by a trained technician in the relative comfort of a laboratory. 
• Spectrophotometers measure individual samples that are prepared and typically placed in a standard size 12.5 mm square glass cuvette before being inserted into the measurement path of the instrument.
• An offline sample-based measurement methodology operates, in nearly all cases, laboratory spectrophotometers. Feedback from measurements taken cannot be considered real-time.

Validation Basics

Two methods: 

1. Vary process concentration

The best way to validate a process photometer is to vary the process concentration itself between certain maxima and minima, taking samples at cardinal points and comparing the process photometer reading to laboratory data. 

Potential con: This may not be practical for a continuously running production system. 

1. Remove in-line process photometer measurement

An alternative method would be to remove the in-line process photometer measurement cell from the process line and introduce a series of verified samples or standard solutions in a controlled manner to confirm agreement. 

Potential con: This method is not always desirable as the process must be interrupted and it may not be possible where line fluids are potentially hazardous to health or present disposal and other challenges. Both of these methods require process disruption and a considerable labor overhead.

Wavelength Validation

Validation of the wavelength accuracy of a spectrophotometer is undertaken using a liquid sample containing a number of distinct peaks such as a solution of holmium perchlorate or a holmium oxide and/or didymium doped glass filter.  Emission peaks enable some spectrophotometers to automatically calibrate the instrument, offer methods to validate the wavelength, and automatically generate a validation report.

Solid glass type validation filters provide a convenient and safe method to quickly validate wavelength accuracy. However, it is not possible to validate wavelength accuracy of a filter photometer using wavelength calibration standards. Most filter photometers operate at one or two fixed wavelengths and are therefore not able to resolve the distinct peaks that the wavelength validation standard provides.

For filter photometer designs that utilize fiber optic connections to the inline measurement cell, a portable fiber optic type spectrophotometer can validate wavelength accuracy. 

Photometric Performance Validation

Let’s look briefly at three methods for validating photometric performance: 

1. Process samples
2. Certified optical filters
3. Certified standards

1. Process Samples

This is probably the most typical validation method employed in the field. A sample of the process stream is taken along with a simultaneous reading of the photometer output on the instrument display. The process sample is analyzed offline and the photometer reading is compared to the result of the analysis. 

Potential con: This method is limited to only one sample concentration and may not provide a true indication of how the instrument will perform under changing process conditions

2. Certified Optical Filters

Certified filters are used to validate that a filter photometer is working to its absorbance specification.  NIST have specified a range of robust glass type validation filters that are suitable for use with both laboratory spectrophotometers and field process photometers that use a standard 12.5mm type square cuvette filter holder.

This type of non-intrusive traceable reference standard verifies photometric accuracy and linearity, both of which are critical to measurement result quality. 

Pros: The glass type validation filters specified by NIST exhibit long term stability and are simple and convenient to use. Metal on fused silica glass filters exhibit a relatively flat transmission profile over a wide wavelength range of 250nm-2200nm, compared to the neutral density type filters that are limited to 360-1100nm (do check the filter is suitable for the instrument under test). 

Note: Process photometers that can be verified without the need to interfere with the process line allow regular trouble-free validation, assuring confidence while saving valuable time and resources.

3. Certified liquid standards

Liquid standards are commercially available to calibrate and validate a wide variety of optical instrument measurement units. They can be introduced into a photometer light path in one of two ways: Directly into the sample cell or using standard cuvettes or proprietary insertable liquid holding devices. 

Pro: Although messy, using liquid solutions is specific

Con: Liquid Standards can exhibit quite wide variability in value, so care must be taken when using them..

Summing up: The pros of photometer validation and verification methods

• Reduced production costs: Filter type photometers provide valuable real-time measurements into an overall control scheme and assist in reducing production costs by improving product consistency and quality. 
• Enhanced measurement accuracy: An understanding of the stray light issues surrounding filter-based photometers can greatly improve the approach to field validation and can enhance measurement accuracy. 
• Improved confidence: The ability to use photometric standards certified by third parties to nationally maintained standards for measurement and performance validation allows a transparent correlation between offline and online measurements and therefore provides a greater confidence in the results provided by inline photometers. 
• Enabling of corrective action: Using multiple validation points can help describe non-linearity exhibited by a photometer and allow action to be taken to correct it. The use of filters and absorbance standards to validate photometers removes the need to take photometers offline and handle potentially hazardous materials as test samples. 

Do you have questions for us about photometers, or other measurement instrumentation and analyzer solutions? Please contact us. Our experts stand ready to assist.

One of the primary concerns when using an inline process analyzer for real-time liquid or gas analysis is validating the instrument is working to specification. Process photometers are often calibrated using liquid or gas calibration standards or directly to the process. These calibration methods can require considerable resources, so the preferred maintenance approach is often to validate performance as opposed to full recalibration of the instrument.

The most typical validation method is verification of instantaneous reported instrument reading against process sample measured offline. This method is limited to only one sample concentration at a time and may not provide a true indication of how the instrument will perform under changing process conditions. This paper will discuss the pros and cons of various photometer validation and verification methods and the impact photometer instrument design has on these methods.

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