WHITEPAPER - Shimadzu · 2018. 1. 12. · Data3:STD 60°C.lcd Detector Data2:STD 70°C.lcd Detector...

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Key Parameter ConceptsPart 1 - Effect of Column Temperature on Bioethanol High Performance Liquid ChromatographyJames Mott, Ph.D., Shimadzu Scientific Instruments, Inc.

Analytical and Measuring Instruments

In the course of performing fermentation monitoring by high performance liquid chromatography (HPLC), bioethanol plant laboratories are using methods that may have been set up by instrument manufacturers or installers, based on a set of operational parameters that “seem appropriate”. How do these labs know if the method parameters they are using are truly the best parameters for securing the most reliable data from their analyses? Certainly, many of the parameters are appropriate based on the recommendations from the column manufacturer, but what does that mean in the actual performance of the HPLC analysis? This article looks at the results of a simple study conducted to investigate the effect of different column temperatures on the resulting chromatograms. The ion exclusion form of chromatography commonly used in fermentation monitoring is a combination of several different mechanisms, all of which may be affected by temperature. Increasing column temperature will have an effect on the mobile phase viscosity, but also will increase the kinetic mobility of molecules in solution, thereby enhancing or enabling the occurrence of interactions. The new Shimadzu i-Series Bioethanol HPLC Analyzer, a compact HPLC system with a high performance autosampler and built-in column heater, was used for the analysis. For peak detection, the new RID-20A Refractive Index Detector was used, and the Lab Solutions LC software (Ver. 5.82) was used for data processing. The Phenomenex Rezex ROA (7.8 x 300 mm) column was used with a Security Guard cartridge to protect the analytical column from unwanted cationic intrusion. The manufacturer’s recommended conditions for this column are a 0.600 mL/min flow rate, a maximum operating pressure of 1000 psi, and a maximum column temperature of 80°C. For this study, a flow rate of 0.600 mL/min was used with a 5 µL sample injection. The detector flow cell was maintained at 40°C, and data was collected at 5 Hz. The mobile phase used was 0.005 N sulfuric acid obtained from Chata Biosystems. The column temperature was varied from 40°C to 80°C, in 10°C increments. Two injections of a standard were made at each temperature, followed by a single injection of a fermentation sample before changing to the next temperature setting. This allowed the column to fully equilibrate at the set temperature. For comparison purposes, only the last standard of the set of two was used, along with the fermentation sample. ■ System Pressure The backpressure measured at the pump pressure transducer will change with the column temperature, so increasing the column temperature will reduce the viscosity of the mobile phase. This in turn will reduce the pressure on the system. In this study, the system pressure dropped nearly 200 psi as the temperature rose from 40°C to 80°C.

Table 1: System Backpressure vs Column Temperature. The reduced pressure due to lower mobile phase viscosity also means that there is less resistance to movement of the sample molecules in solution while allowing molecules to interact more readily with the column matrix to affect separation. The question, however, is whether this change in column temperature makes that much of a difference. ■ Key Parameters To assess the effect that temperature has on the chromatographic separation of the standard components being monitored during this analysis, there are certain observable parameters that may give insight into the optimum

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temperature. In this analysis, the parameters selected were: Retention Time, USP Width, Area/Height Ratio, Tailing, Theoretical Plate Number, Resolution, and Signal/Noise Ratio. Retention Time: This will indicate if temperature has an effect on the relative retention time of the components in the standard mixture. If there is an observable effect, the question becomes whether or not all of the analytical components are affected to the same degree. USP Width: This is a measure of the theoretical width of a peak at the baseline of the peak, which is determined by setting tangent lines at the inflection points along the sides of the peaks, and then extending those tangent lines to the baseline. The USP width is determined by simply subtracting the peak start time from the peak end time, as defined by the time values where the tangent lines meet the baseline. In chromatography, narrow peaks are considered to be better than wide peaks, as they tend to be taller and can be distinguished and identified more easily, thereby providing better measurement results. Area/Height Ratio: This is simply the ratio of the peak area to the peak height. More importantly, it is an approximation of the width of the peak at one-half of its height. If the area is measured in units of µV x seconds, and the height is measured in µV, then the ratio will be in seconds. As with the USP Width, the narrower the peak, the better. Tailing Factor: A ratio of the total peak width time at 5% of the peak height to twice the “front half” width (the time difference from the peak start point at 5% of peak height to the apex time). A perfectly symmetrical peak shape would have a Tailing Factor of 1.00. The closer to 1.00, the better.

T = Peak Tailing Factor W 0.05 = The distance from the leading edge to the tailing edge of the peak, measured at a point equal to 5% of the peak height from the baseline. f = The distance from the peak apex to the leading edge of the peak measured at a height from the baseline corresponding to 5% of the peak height.

Theoretical Plate Number: This is a number that provides a conceptual value to the number of partitioning steps that occur for each analyte as it moves through the HPLC column. The value is calculated from the retention time of the peak and its width. In general, the number of theoretical plates will increase for peaks that elute later in the chromatogram. Also, as a generality, anything that can be done to make peaks narrower will increase the theoretical plate number.

N = Theoretical Plate Number t = Peak Retention Time W = Peak Width at Base

Resolution: The resolution value is a judgement of how well a peak is separated from the peak immediately before it in the chromatogram. As would be expected, the first peak, not having a peak before it, would have a resolution value of 0.00. Common HPLC ideology states that a resolution value of ≈1.5 should be a full baseline separation (the end of the preceding peak and the start of the subsequent peak are at the chromatographic true baseline.

R = Resolution between an integrated peak of interest (Peak 2) and the peak preceding it (Peak 1). t2 = The measured retention time of Peak 2 t1 = The measured retention time of Peak 1 W2 = The width at the base of Peak 2 W1 = The width at the base of Peak 1

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Signal/Noise Ratio: The ratio of the maximum peak intensity (peak apex intensity) to the average baseline noise from a selected segment of baseline. The higher the signal/noise ratio, the better the limit of detection and quantitation. ■ The Effect of Temperature on a Standard Sample Retention Time The overlaid chromatograms below are of the same standard sample injected at 5 different column temperatures, ranging from 40°C to 80°C in 10°C increments. In this view, it is quite clear that the column temperature has almost no effect on the maltose peak, and very little effect of the glycerol peak. However, the obvious shifting of the acetic acid peak indicates that acetic acid retention is most significantly affected by the change in column temperature.

Fermentation Calibration Standard Mix at 5 Different Column Temperatures By applying a base shift to these 5 chromatograms, it is easier to see the relative differences caused by the change in column temperature. Looking at the segment of the chromatogram that represents the DP4+ to Glucose peaks, it is obvious that increasing column temperature causes the DP4+ and DP3 peaks shift slightly toward earlier retention times, that maltose has a very stable retention time over the temperature range, and glucose shifts slightly toward later retention times.

Segment of Chromatograms (DP4+, DP3, Maltose, and Glucose) at 5 Different Temperatures Focusing on the middle part of the chromatogram, it can be seen that the retention time of glycerol initially shifts to later times, and then appears to start moving back towards an earlier time. Lactic acid and acetic acid both move toward earlier elution times. This has the effect of increasing the separation space between lactic acid and glycerol, while decreasing the space between glycerol and acetic acid.

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Segment of Chromatograms (Lactic Acid, Glycerol, and Acetic Acid) at 5 Different Temperatures Lastly, the ethanol elution peak is affected by increasing temperature much like glycerol, but to a more significant extent. The peak initially shifts to a later time, and then reverses that tendency due to earlier elution.

Segment of Chromatograms (Ethanol) at 5 Different Temperatures

Name 40°C 50°C 60°C 70°C 80°C DP4+ 6.784 6.741 6.696 6.65 6.602 DP3 7.65 7.627 7.606 7.58 7.551 Maltose 8.412 8.418 8.426 8.426 8.421 Glucose 10.001 10.065 10.129 10.18 10.221 Lactic Acid 13.833 13.785 13.716 13.619 13.501 Glycerol 14.467 14.523 14.56 14.566 14.548 Acetic Acid 16.432 16.251 16.055 15.837 15.604 Ethanol 22.274 22.64 22.84 22.874 22.771

Table 2: Relationship between Peak Retention Time and Column Temperature USP Width It can be difficult to determine if peak width has changed using simple visual inspection of the chromatograms. Fortunately, the data acquisition software system can automatically determine the USP Width for all integrated peaks, and provide values for these peaks, respectively. In many forms of chromatography, increasing the column temperature will result in narrower peak shapes, as the mobile phase becomes less viscous and presents less physical resistance to the movement of molecules through the mobile phase. This allows for more efficient interactions of the analyte molecules with the stationary phase

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(column). However, in the data shown below, the effect of increasing temperature on the ion exclusion separation of these standard materials is not as consistent. As can be seen, the first four standard peaks (all carbohydrates) show a consistent reduction in the peak width as the column temperature increases. This bodes well for the improvement of the separation of these peaks. But, the last four peaks (organic acids and alcohols) all display increased USP Width with increasing column temperature. Lactic acid has an anomalous value at 40°C due to a small unresolved peak on the leading edge of the main peak. In most cases, this increase in width is quite modest, but the peak with the largest width increase over the temperature range is ethanol.

Name 40°C 50°C 60°C 70°C 80°C DP4+ 0.344 0.343 0.339 0.334 0.332 DP3 0.335 0.314 0.3 0.291 0.285 Maltose 0.33 0.313 0.301 0.293 0.29 Glucose 0.349 0.328 0.317 0.313 0.313 Lactic Acid 0.411 0.395 0.404 0.412 0.413 Glycerol 0.408 0.409 0.415 0.423 0.43 Acetic Acid 0.452 0.453 0.465 0.472 0.482 Ethanol 0.627 0.655 0.685 0.714 0.741

Table 3: Relationship between Peak USP Width and Column Temperature

Plot of USP Peak Widths for Fermentation Calibration Standard Peaks at Five Temperatures Area/Height Ratio While the USP Width tends to show the peak width extrapolated to the baseline, the Area/Height (A/H) ratio approximates the width of the peak at about 50% of its height. This can be useful for setting integration parameters, but it also tends to show peak width without as much influence due to tailing of the peak. As with the USP Width, lactic acid has a much larger A/H ratio at 40°C due to the influence of a non-resolved shoulder on the leading edge of the peak.

Name 40°C 50°C 60°C 70°C 80°C DP4+ 13.975 13.929 13.819 13.621 13.595 DP3 13.448 12.801 12.27 11.987 11.959 Maltose 13.204 12.529 12.059 11.829 11.854 Glucose 13.807 13 12.557 12.427 12.428 Lactic Acid 18.221 15.405 16.216 16.925 16.573 Glycerol 16.481 16.329 16.828 16.93 16.88 Acetic Acid 17.877 17.696 18.989 19.059 18.802 Ethanol 25.326 26.17 27.125 28.072 28.842

Table 4: Relationship between A/H Ratio and Column Temperature

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Plot of Area/Height Ratio for Fermentation Calibration Standard Peaks at 5 Different Temperatures Tailing Factor

Name 40°C 50°C 60°C 70°C 80°C DP4+ 1.267 1.273 1.273 1.263 1.258 DP3 1.117 1.133 1.146 1.156 1.179 Maltose 1.095 1.112 1.121 1.125 1.134 Glucose 1.065 1.074 1.077 1.077 1.076 Lactic Acid 0 0 0 0.936 0.954 Glycerol 1.005 1.02 1.029 1.032 1.034 Acetic Acid 0.975 0.997 0.992 1.002 1.042 Ethanol 1.053 1.054 1.06 1.065 1.07

Table 5: Relationship Between Tailing Factor and Column Temperature

Plot of Tailing Factor for Fermentation Calibration Standard Peaks at 5 Different Temperatures

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Name 40°C 50°C 60°C 70°C 80°C DP4+ 6222 6182 6247 6332 6327 DP3 8355 9413 10302 10888 11195 Maltose 10376 11602 12576 13197 13464 Glucose 13166 15112 16340 16878 17039 Lactic Acid 18084 19489 18452 17498 17062 Glycerol 20128 20190 19670 18987 18342 Acetic Acid 21159 20575 19115 18023 16766 Ethanol 20163 19122 17791 16405 15106

Table 6: Relationship Between Theoretical Plate Number and Column Temperature

Plot of Theoretical Plate Number for Fermentation Calibration Standard Peaks at 5 Different Temperatures Resolution By definition, resolution is measured as the separation of a peak from the peak ahead of it. The first peak cannot have a value.

Name 40°C 50°C 60°C 70°C 80°C DP4+ -- -- -- -- -- DP3 2.552 2.697 2.848 2.976 3.073 Maltose 2.29 2.522 2.732 2.898 3.025 Glucose 0.927 1.031 1.333 1.381 1.213 Lactic Acid 2.16 0.812 1.022 1.09 1.171 Glycerol 1.548 1.837 2.059 2.268 2.484 Acetic Acid 4.571 4.01 3.399 2.84 2.317 Ethanol 10.825 11.532 9.372 11.866 11.719

Table 7: The Relationship between Chromatographic Resolution and Column Temperature.

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Plot of Resolution Value for Fermentation Calibration Standard Peaks at 5 Different Temperatures. Signal-to-Noise Ratio For this measurement, the signal intensity (peak height) to average noise ratio was taken in the time segments of 3.0 – 5.5 minutes of the chromatogram. This noise segment is before any peaks should elute from the standard sample.

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Maltose 2457 2536 2958 3331 3016

Glucose 4680 4888 5679 6401 5985

Lactic Acid 156 152 179 204 190

Glycerol 796 773 867 953 867

Acetic Acid 165 163 185 205 189

Ethanol 2652 2530 2752 2967 2690

Table 8: Relationship between Signal-to-Noise Ratio and Column Temperature

Plot of Signal-to-Noise Ratio for Fermentation Calibration Standard Peaks at 5 Different Temperatures

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■ Conclusions from Standard Chromatograms The results of the temperature experiments are largely inconclusive for many of the key parameters. In many cases, the values deemed “better” for the first four peaks occurred at high temperature, while the “better” values for the last four peaks trended toward the lower temperatures. Obviously, this is not helpful in trying to determine an optimal analytical column temperature. This forces some decisions to be made by the laboratory. The numbers from the analysis of the standard would seem to indicate that there isn’t a clear choice for column temperature. An experienced chromatographer might look at these key parameters and exclude from consideration those that do not seem to have a strong correlation to improving the analytical result. That is, those parameters that tend to be most important are Resolution and Signal-to-Noise Ratio, as they can make the case for “best separation” and “best sensitivity”. In looking at the standard chromatograms run at different temperatures, there is a very slight improvement in the separation of the first four peaks with increasing temperature. More significant is the temperature effect on the separation of the Lactic Acid – Glycerol – Acetic Acid trio. The two organic acid peaks are the most affected by the temperature change. Clearly, the separation of Lactic Acid, relative to Glycerol, is better in the 60 – 80°C temperature range. Acetic Acid is far better separated from Glycerol at 40°C, but still has very adequate separation at higher temperatures. This may lead to the conclusion that a column temperature of 70°C may be best for overall performance of the analysis. Lastly, looking at the maximum Signal-to-Noise ratios, it must be concluded that the sensitivity is best when the column temperature is 70°C. ■ The Effect of Temperature on a Fermentation Sample All of the previous data was performed by running the same standard sample at different temperature. It is worthy to note that the analytical method is very repeatable.

Overlay of 6 Replicate Injections of the Same Standard with 70°C Column Temperature.

Sample Name DP4+ DP3 Maltose Glucose Lactic Acid Glycerol Acetic Acid Ethanol STD Repeat001 6.648 7.579 8.424 10.177 13.624 14.565 15.838 22.819 STD Repeat002 6.646 7.576 8.421 10.173 13.620 14.560 15.834 22.812 STD Repeat003 6.647 7.578 8.424 10.176 13.624 14.564 15.838 22.819 STD Repeat004 6.644 7.577 8.423 10.177 13.626 14.566 15.840 22.820 STD Repeat005 6.643 7.574 8.419 10.171 13.618 14.557 15.831 22.809 STD Repeat006 6.641 7.572 8.417 10.170 13.616 14.556 15.830 22.806 AVERAGE 6.645 7.576 8.421 10.174 13.622 14.561 15.835 22.814 %RSD 0.037 0.033 0.032 0.032 0.029 0.030 0.027 0.026

Table 9: Retention Times for 6 Replicate Standard Injections The finding that the retention times of “known” substances is very stable is very important when comparing the retention times of peaks found in fermentation broth samples. These samples are much more complex, having many additional substances beyond the 8 analytes normally being tracked in the fermentation. While 70°C seems

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to be a good “compromise” column temperature for analysis of standards, the question is how temperature may affect a more complex chromatogram resulting from a fermentation sample? The analysis of a fermentation sample at the same temperature range (40 °C – 80°C) as used with the standards generates some interesting results.

Fermentation Sample Analyzed at 5 Different Column Temperatures. As was seen with the chromatograms of the standard sample, the easily identifiable peaks shift in the same way. Glucose shifts to a slightly later retention time, glycerol moves very little, and ethanol still has that same tendency to move, at first, to a later retention time, and then starts moving earlier again as the temperature rises. Even Lactic Acid (blue arrow) and Acetic Acid (green arrow) move as was seen in the standard:

11.6 – 17.5 Minute Segment; Fermentation Sample Analyzed at 5 Different Column Temperatures. More interesting findings are located at the front part of the fermentation sample chromatogram, however. Looking at the 6 – 10 minute time segment (see below), it is clear that there is much more complexity than is found with the standard sample, and peak identities can be a bit confusing. Easily noticed is that the DP4+ area (blue arrow) becomes more split as the column temperature is increased. This is often a concern in many bioethanol labs, as the processing of this area into a single group or peak area is necessary to obtain a truly representative measurement. This also shows that the chemical nature of the DP4+ peak changes over the course of the fermentation, and the column performance may give significantly different degrees of separation. In the area where Maltose should elute (red arrow), there are many competing materials that can also elute in the same time range. At 40°C, all of these materials partially coalesce into one poorly shaped peak. As the column temperature increases, better definition of the individual material peaks becomes apparent, with a semi-triangular peak (dashed line) moving away from this area. But, where is Maltose?

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6.0 – 10.0 Minute Segment, Fermentation Sample Analyzed at 5 Different Column Temperatures. The overlay shown below is that of the fermentation sample and the calibration standard, both analyzed with a 70°C column temperature. We know from the prior evidence that the peak retention times for the known peaks are very repeatable, so overlaying the standard should help to identify the peak locations in the fermentation sample. The alignment of the Maltose peak from the standard (black arrow) with the corresponding hump in the fermentation sample should be good evidence where maltose is in the fermentation sample. Also of interest is that the DP3 peak of the standard (red arrow) does not line up well with any peak in the fermentation sample. Is this an indication that this fermentation sample does not contain any DP3? If so, what is that mystery peak?

Early Chromatogram Segment Comparison, Fermentation Sample vs Standard Sample at 70°C ■ Conclusions from Fermentation Sample Chromatograms It is clear that column temperature has a highly significant effect on the elution of the many peaks that can be present in a fermentation sample. With the aim of attaining a clean value for some of the peaks of interest, such as Maltose, higher temperature aids in getting a discernable peak for Maltose in samples where other competing materials may generate about the same peak intensity. Of the column temperatures tested, the 6 – 10 range of the chromatogram appears to give the best results at 70°C or 80°C. However, as seen with the standard sample, Acetic Acid is slightly better separated from Glycerol at 70°C.

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The final conclusion, therefore, is that the chromatography, as produced on this test HPLC system with this specific column, is that 70°C appears to be the best column temperature for overall performance. Analytical columns from different lots from the same manufacturer or from different manufacturers may have slightly different results. If a lab is currently running at 65°C column temperature, is it worthwhile to reset the column to 70°C? That is a decision that must be left up to the lab manager, but it might help with the ability to get a more reproducible integration of the Maltose peak in certain fermentation samples. ■ Future Studies

• Part 2 – How Does System Flow Rate Affect the Chromatography of Fermentation Sample? • Part 3 – What Effect Does Column Dimension Have on the Chromatography of Fermentation Samples? • Part 4 – What Effect Does the Mobile Phase Sulfuric Acid Concentration Have on the Chromatography of

Fermentation Sample?

For Research Use Only. Not for use in diagnostic procedures. The contents of this publication are provided to you “as is” without warranty of any kind, and are subject to change without notice. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication.

SHIMADZU Corporation www.shimadzu.com/an/

Second Edition: January 2017

© Shimadzu Corporation, 2017

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WHITEPAPER - Shimadzu · 2018. 1. 12. · Data3:STD 60°C.lcd Detector Data2:STD 70°C.lcd Detector...

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