BACKGROUND
a popular liquid chromatography column used for the analysis of biomass conversion and fermentation products is based on a polymer matrix of polystyrene-divinylbenzene  .
it is operated in isocratic elution mode enabling the connection to a refractive index detector  which provides universal compound detection.
this set up allows for analysis of a wide range of compounds.
with a standard 300 mm length, this type of column provides baseline separation of the main cell wall sugars  of many lignocellulosic biomass feedstocks.
this column is also used for the analysis of organic acids, alcohols  and sugar degradation products  .
it only requires acidified water as mobile phase and only minimal sample preparation.
typical hplc methods aim to separate complex mixtures in order to individually detect isolated compounds – particularly when a non-specific detector like rid is used.
in our experience from supporting researchers in an organization comprising more than  <dig> scientists dedicated to bioenergy research, many analytical questions can be reduced to identifying the change in a few compounds over the course of the experiment.
furthermore, reducing analysis time is often more important than obtaining precise absolute quantification data.
this is especially true for the results from screening experiments involving a larger amount of samples.
the detection of relative changes is often sufficient to identify promising candidates for further analysis.
fast methods targeting individual compounds in complex mixtures have previously been established and include, for example, gas chromatography  <cit>  spectroscopy  <cit> , or enzymatic assays, such as for sugars, sugar alcohols, organic acids, and ethanol .
several of these target analytes can even be measured in an automated mode using a biochemistry analyzer  and results are obtained in minutes  <cit> .
however, all such methods either require costly equipment or individual analysis kits for every single analyte.
therefore, it is tempting to use existing analytical instrumentation  and reduce analysis time for higher sample throughput.
in this respect, scarlata and hyman  <cit>  have successfully shown that reducing the column length to 100 mm  can reduce the analysis time by a factor of five compared to the 300 mm column, but still provide adequate accurate results from a biomass compositional analysis .
in our studies, we explored a further reduction of the analysis time by using only a guard column as the simplest and most basic column available.
for this purpose, samples generated by various research groups in our institute were analyzed on the standard 300 mm aminex hpx-87h column and compared to results obtained from an analysis on a guard column .
RESULTS
a short column length is often used for faster separation especially in conjunction with mass spectrometric detection.
these shorter columns have usually smaller particle sizes  which greatly improve resolution.
however, columns like the aminex hpx-87h operate in a mixture of size- and ion-exchange/exclusion.
they consist of a resin with a rather large particle size .
as a result, a reduction in column length significantly lowers column efficiency.
for example, a reduction of the column length to 50 mm results in practically unresolved glucose and xylose .
these types of columns can therefore not be directly compared to other columns.
despite the lower separation efficiency, the shorter columns can still provide adequate chromatographic resolution when used with appropriate samples in which compounds co-eluting with the target analytes are either not present or their concentration is sufficiently low so they do not significantly interfere with quantification.
figure  <dig> shows the elution profile of some of the most common fermentation products  and sugar degradation products , furfural) on the 50 mm rezex rfq fast acid h+ guard column  operated at different temperatures at a flow rate of  <dig>  ml/min.
the degradation products 5-hmf and furfural are promising precursors for the generation of biofuels and value-added chemicals  <cit> .
good separation of all these compounds is achieved at a column temperature of  <dig> °c .
at this temperature, ethanol  and acetone  are almost baseline separated.
by increasing the column temperature the alcohols elute later and 5-hmf  and furfural  elute earlier.
the retention time of acetone  is practically not affected by temperature .
ethanol/acetone  separation is deteriorated at a higher temperature and 5-hmf  is co-eluting with n-butanol  at  <dig> °c  and with iso-butanol  at  <dig> °c .
the column temperature must therefore be chosen based on the sample composition so that target analytes do not overlap.
for example, as mentioned above, a temperature of  <dig> °c has to be applied for successful separation of ethanol and acetone.
this column temperature is also beneficial for the faster elution of iso-and n-butanol .
in contrast, 5-hmf and furfural are eluted faster at a higher temperature.
however, depending on which butanol isomer is present, the column temperature needs to be correctly chosen since 5-hmf coelutes either with n-butanol at  <dig> °c  or iso-butanol at  <dig> °c .fig.
<dig> separation of ethanol , acetone , iso-butanol , n-butanol , 5-hmf  and furfural  on the 50 mm guard column at a column temperature of   <dig> °c,   <dig> °c and   <dig> °c.hplc parameters: 50 mm ×  <dig>  mm rfq fast acid h+ column, mobile phase 5 mm sulfuric acid, flow rate  <dig>  ml/min, refractive index detection
using a 20 μl injection volume, the determined linear calibrations for the compounds under study ranged from  <dig> - <dig>  mg/ml to 50 mg/ml  with ri and uv detection .
uv detection  resulted in an about 100-fold increased sensitivity for 5-hmf and furfural compared to ri detection .table  <dig> calibration results for each compound
chromatography conditions: 50 mm ×  <dig>  mm rfq fast acid h+ kept at  <dig> °c, flow rate:  <dig>  ml/min of 5 mm sulfuric acid, 20 μl injection volume; lower limit of the calibration range was determined as the concentration level with a relative standard deviation  <10 %
since  <dig> °c is close to the maximum operating temperature of the column , we chose a column temperature of  <dig> °c for most of the following experiments.
this is good balance between fast analysis time and column life time.
application to analysis of n-butanol and iso-butanol production during fermentations
these two alcohols elute later than most other components present in the fermentation media.
fig.
2a shows a representative chromatogram from an e.coli fermentation producing n-butanol  which eluted at  <dig>  min.
fig.
2b shows the determined n-butanol production of three e. coli strains after incubation for 24 h, 48 h and 72 h. the n-butanol profile obtained with the 50 mm guard column was very similar compared to the 300 mm bio-rad hpx-87h column .
values reached  <dig> - <dig>  % of the values obtained with the longer column .
the dh <dig> strain is able to produce more n-butanol than bw <dig>  which almost showed no significant n-butanol levels.
usually, bw <dig> produces about half the concentration of dh <dig> .
unexpectedly, the bw <dig> strain used in this experiment did not perform as expected.
the deletion of acrb efflux pumps in dh1Δacrb lead to even higher n-butanol levels.
acrb pumps are large proteins and are used to actively secret n-butanol from the cell.
however, they can be considered “energy drainers” since they use a big portion of the cellular resources needed for n-butanol synthesis.
at the levels measured here, n-butanol is not toxic for the e. coli cells and therefore the acrb pumps present in the dh <dig> strain are not a “de-toxification”-advantage over the dh1Δacrb strain which has more energy resources available for n-butanol production.fig.
<dig> representative chromatogram of the supernatant from escherichia coli strain dh <dig> fermentation analyzed with  the 50 mm guard column showing n-butanol  elution, and  n-butanol concentration of the supernatant from e. coli strains dh <dig>  dh1Δacrb and bw <dig> fermentations after 24 h, 48 h and 72 h analyzed with the 50 mm guard column, 300 mm column and 30 mm guard column.
hplc parameters: column temperature 55 °c, mobile phase 5 mm sulfuric acid, flow rate  <dig>  ml/min , refractive index detection
a similar chromatogram was obtained for iso-butanol production from e. coli strains sfab <dig> and sfab <dig> .
iso-butanol  eluted at  <dig>  min in a region where no interference from other media components were expected.
the values measured with the 50 mm guard column were again almost identical to the ones obtained with the 300 mm column .
in this experiment, the decrease of iso-butanol after 48 h for the sfab  <dig> strain was explained by evaporation due to a leak in the cap of the flask.fig.
<dig> representative chromatogram of the supernatant from escherichia coli strain sfab <dig> fermentation analyzed with  the 50 mm guard column showing iso-butanol  elution, and  iso-butanol concentration profile of the supernatant from e. coli strains sfab <dig> and sfab <dig> fermentation between 24 h and 192 h analyzed with the 50 mm guard column and 300 mm column.
hplc parameters: column temperature 55 °c, mobile phase 5 mm sulfuric acid, flow rate  <dig>  ml/min , refractive index detection
the analysis of n- and iso-butanol was performed with a 4 min total run time and this is about 9– <dig> times faster compared to the standard 300 mm column.
application to analysis of 5-hydroxymethylfurfural  and furfural
5-hmf and especially furfural are compounds with long elution times on the standard 300 mm hpx-87h column .
experiments requiring the analysis of these compounds often include determination of the degree of their removal or reduction during membrane detoxification of hydrolysate  <cit>  or the rate of sugar degradation during pretreatment or conversion steps  <cit> .
for the purpose of this study, three biomass hydrolysates prepared under different severity conditions were analyzed.
fig.
4a depicts the elution profiles for the two compounds on the 50 mm guard column.
hydrolysate  <dig> was prepared with a lower sulfuric acid concentration  and incubated at a lower temperature  causing less sugar degradation.
therefore, it shows a lower 5-hmf  and a lower furfural  concentration compared to the other two hydrolysates which were prepared with a higher acid concentration  and a higher temperature .
as illustrated in fig.
4b, the 5-hmf analysis results with the 50 mm guard column compared to the 300 mm column were lower for hydrolysate  <dig> , lower for hydrolysate  <dig>  and lower for hydrolysate  <dig> .
however, the furfural amounts determined with the 50 mm guard column were in the range of  <dig>  %- <dig>  % of the 300 mm column results  and can be considered very comparable.
in conclusion, the different pretreatment severity profiles were adequately reflected by analysis with the 50 mm guard column.fig.
<dig> chromatogram of hydrolysate  <dig>  hydrolysate  <dig> and hydrolysate  <dig> analyzed with  the 50 mm guard column showing 5-hmf  and furfural  elution, and  5-hmf and furfural concentrations of hydrolysate  <dig>  hydrolysate  <dig> and hydrolysate  <dig> analyzed with the 50 mm guard column, 300 mm column and 30 mm guard column.
hplc parameters: column temperature  <dig> °c, mobile phase 5 mm sulfuric acid, flow rate  <dig>  ml/min , refractive index detection
application to analysis of ethanol production and xylose and glucose consumption during fermentations
when glucose and xylose are present in a mixture, their accurate determination using the 50 mm guard column is challenging since the separation of these two sugars is practically not possible.
however, the analysis can be performed when either one of the two is present in high abundance or one of the two monosaccharides concentrations is constant over the course of the experiment.
ethanol eluted at  <dig>  min which was close to other fermentation media compounds but was still sufficiently isolated for selective measurement in these experiments .fig.
<dig> representative chromatogram of the supernatant from a saccharomyces cerevisiae sa-1-x <dig> strain fermentation analyzed with  the 50 mm guard column showing xylose  and ethanol  elution, and  xylose and ethanol concentration profile of the supernatant from a modified s. cerevisiae strain fermentation between 0 h and 62 h analyzed with the 50 mm guard column and 300 mm column.
hplc parameters: column temperature 55 °c, mobile phase 5 mm sulfuric acid, flow rate  <dig>  ml/min , refractive index detection
fig.
<dig> chromatogram of the supernatant from saccharomyces cerevisiae sa- <dig> strain fermentation supplemented with 25 % of ph adjusted hydrolysate  <dig> analyzed with the 50 mm guard column showing glucose , xylose  and ethanol  elution after  42 h,  50 h and  67 h, and xylose and ethanol concentration profile of the supernatant between 0 h and 62 h from a s. cerevisiae sa- <dig> strain fermentation supplemented with  25 % of pervaporation detoxified hydrolysate  <dig> and  25 % of ph adjusted hydrolysate  <dig>  analyzed with the 50 mm guard column and 300 mm column.
hplc parameters: column temperature  <dig> °c, mobile phase 5 mm sulfuric acid, flow rate  <dig>  ml/min , refractive index detection
the consumption of xylose and production of ethanol of a modified saccharomyces cerevisiae strain analyzed with the 50 mm guard column and 300 mm column is shown in fig.
5b and a representative chromatogram is shown in fig.
5a.
the xylose amounts measured with the 50 mm guard column were in the range of  <dig> - <dig>  % and the ethanol amounts were in the range of  <dig> - <dig>  %  of the values obtained with the 300 mm column.
the two lower concentrations of ethanol showed  <dig>  %  and  <dig>  %  deviation most likely due to interfering co-eluting compounds at these lower concentrations.
overall, the consumption/production profile measured with the 50 mm guard column is very similar to the one obtained with the 300 mm column.
however, the total run time using the 50 mm guard column was only 3 min and ethanol elution was completed at  <dig>  min.
this is about  <dig> times faster compared to the 300 mm column.
in another experiment, s. cerevisiae sa- <dig> was grown in a medium supplemented with 25 % hydrolysate  <dig> that was either ph adjusted or detoxified by membrane pervaporation which reduced the concentration of toxic compounds such as furfural, formic and acetic acid  <cit> .
the hydrolysate was obtained by a dilute acid and heat pretreatment of miscanthus biomass in order to release hemicellulosic sugars and improve cellulose digestibility.
during this process, the harsh conditions lead to sugar and lignin degradation resulting in the production of a fleet of fermentation inhibitors like furans, phenolics and organic acids which could also interfere with the analysis  <cit> .
representative chromatograms of the sample with ph-adjusted hydrolysate at time points 42 h, 50 h and 67 h analyzed with the 50 mm guard column are shown in fig.
6a-c. a decrease of glucose  and increase of ethanol  can be observed over the time course whereas the remaining chromatogram is mostly the same.
the inset in fig.
6a shows the close elution of glucose  and xylose  resulting in a peak with a shoulder making accurate integration difficult.
fig.
6d and 6e show the measured glucose and ethanol concentrations over the course of the experiments with the membrane-detoxified hydrolysate and the ph-adjusted hydrolysate, respectively.
the values for “glucose uncorrected” were obtained when the attempt was made to integrate glucose separately from the closely-eluting xylose peak .
the determined “glucose uncorrected” concentrations for experiments with the ph-adjusted and with the detoxified hydrolysate were  <dig> - <dig>  % and  <dig> - <dig>  %, respectively, of the concentrations obtained when analyzed with the 300 mm column.
this indicates that xylose interfered with a correct glucose area determination.
the ethanol concentrations were almost identical with  <dig> - <dig>  % and  <dig> - <dig>  %, respectively, compared to the results of the 300 mm column.
when glucose and xylose were integrated together as one peak and the area of the xylose peak  of the 67 h time point was subtracted , “glucose corrected” values were obtained which were closer to the results of the 300 mm column .
even without glucose correction, the effect of the toxic hydrolysate on delaying the glucose fermentation and production of ethanol until around 42 h into the fermentation was clearly observed with the 50 mm column .
application to analysis of glucose release from enzymatic digestibility assays of pretreated biomass
the enzymatic release of glucose from cellulose after biomass pretreatment is often performed not only to determine of the effectiveness of the pretreatment itself but also for the evaluation of the hydrolytic potential of new enzymes.
a commonly used method is the pretreatment of biomass with dilute acid  at higher temperatures  which leaves behind a solid cake consisting of mainly lignin and decrystallized cellulose  <cit> .
after washing and/or neutralization this cake is incubated with cellulose-degrading enzymes in either citrate or acetate buffer at ph  <dig> in order to release glucose  <cit> .
as shown in the chromatogram of the supernatant after enzymatic digestion of “pretreated miscanthus biomass” , citrate/citric acid  of the citrate buffer is interfering with glucose  at lower and higher concentrations on the 50 mm guard column.
furthermore, sorbitol  derived from the commercial enzyme mixture  also interferes, but to a much lower extent.
using the 300 mm column, citric acid , glucose  and sorbitol  can be separated .
as observed earlier with the glucose/xylose integration , the attempt to integrate the glucose peak separately  lead to glucan conversions  that deviated by  <dig> - <dig>  % from the ones obtained using the 300 mm column .
for example, glucan conversion after 72 h was  <dig>  %  vs  <dig>  % .
at time point 0 h , the observed glucose peak  was derived from the enzyme mixture used in the experiment.
therefore, this area had to be subtracted as a blank from the glucose area of following time points.
similar as before , if the citric acid and glucose peaks at timepoint 0 h were integrated as one peak and subtracted from following peaks  the values obtained with the 50 mm guard column  were in the range of  <dig> - <dig>  % of the values with the 300 mm column .
the glucan conversion after 72 h was  <dig>  %  vs  <dig>  % .
the elution on the 50 mm guard column is completed after  <dig>  min and is therefore about  <dig> times faster compared to  <dig>  min with the 300 mm column.fig.
<dig> chromatogram of the supernatant from the enzymatic digestion of “pretreated miscanthus biomass” showing citric acid , glucose , sorbitol  and acetic acid  elution after  0 h and 24 h digestion in citrate buffer analyzed with the 50 mm guard column,  0 h and 24 h digestion in citrate buffer analyzed with the 300 mm column,  24 h digestion in acetate buffer analyzed with the 50 mm guard column; and enzymatically released glucan concentration profile of the supernatant from the enzymatic digestion of “pretreated miscanthus biomass”between 0 h and 72 h using  citrate buffer  acetate buffer analyzed with the 50 mm guard column and 300 mm column.
hplc parameters: column temperature  <dig> °c, mobile phase 5 mm sulfuric acid, flow rate  <dig>  ml/min , refractive index detection.
error bars represent +/−  <dig> standard deviation
using acetate buffer, only sorbitol  derived from the commercial enzyme mixture interfered with glucose on the 50 mm guard column  because acetate  eluted later at  <dig>  min.
a “split-peak” integration of glucose  and sorbitol  is sufficient to obtain glucan conversions within  <dig> - <dig>  % of the ones resulting from using the 300 mm column.
the 72 h conversion was  <dig>  %  vs  <dig>  %  and the run time was again about  <dig> times faster  .
it is noteworthy to mention that the acetate buffered samples can also be run with an analysis time of only  <dig>  min as the citrate buffered samples.
the acetate peak  will then be eluted within the first  <dig>  min of the next sample .
furthermore, the sample analysis time of  <dig>  min is fast enough to compete with other higher-throughput glucose methods like using a ysi bioanalyzer for glucose determination.
with the “overlap injection” function of the autosampler, which allows rapid injection of the next sample after a run is completed, about  <dig> samples  can be processed per day.
application to analysis of ethanol, acetone and n-butanol production during acetone-butanol-ethanol  fermentation
the analysis of the products from acetone-butanol-ethanol  fermentation with a short column is challenging since clostridia secret a fleet of other compounds  which can co-elute with the target analytes  <cit> .
the use of an additional uv detector is beneficial for more accurate compound detection and quantification.
fig.
8a shows the chromatogram of a representative sample with ri detection.
ethanol  and n-butanol  have to be analyzed using the unspecific ri detector.
for n-butanol, the concentration profile obtained with the 50 mm guard column is very similar to the 300 mm column , since interfering compounds were absent or in low concentration at this retention time.
the measured n-butanol concentrations were on average  <dig>  % +/−  <dig>  %  of the values obtained with the 300 mm column, except for early time points  due to lower concentrations affecting accuracy, and the 90 h and 112 h time points with  <dig>  % and  <dig>  % deviation.fig.
<dig> chromatogram of the supernatant from a clostridium acetobutylicum abe fermentation after 23 h analyzed with the 50 mm guard column showing ethanol , n-butanol , ethanol interference , acetone interference  and acetone  analyzed with  refractive index detection  uv 210 nm detection  uv 265 nm detection,  uv 285 nm detection, and concentration profile of the supernatant from a chlostridium acetobutylicum abe fermentation of  n-butanol,  acetone,  ethanol, between 0 h and 112 h analyzed with the 50 mm guard column and 300 mm column.
hplc parameters: column temperature  <dig> °c, mobile phase 5 mm sulfuric acid, flow rate  <dig>  ml/min , refractive index and uv  detection
in the 210 nm uv trace of the same sample  a compound  is visible that is interfering with the ethanol determination using ri detection .
however, with the 300 mm column, peak  <dig> and ethanol can be separated .
if ethanol was determined only by ri detection, concentrations were overestimated and the values ranged  <dig> - <dig>  %  of the values obtained with the 300 mm column .
the interfering compound was present early in the fermentation process when ethanol production was zero or very minimal.
therefore, it should be possible to measure the area of peak  <dig> with 210 nm uv detection and correlate it to the area obtained with ri detection.
in this way, the interference of peak  <dig> in rid mode should be reduced by measuring the sum of the area  with rid and subtracting the theoretical “rid area contribution” of peak  <dig> calculated from its 210 nm uv area.
however, we noticed that the low intensity of peak  <dig> in the earlier time points made correlation of uv and rid signal less accurate.
we therefore used an empirical method to determine the uv/rid correlation by adjusting the correlation factor so the corrected ethanol concentration from the 50 mm guard column best matched the 300 mm column results.
this correction factor was applied to the uv-to-rid conversion of peak  <dig> at all time-points of the fermentation.
“ethanol corrected” values of average  <dig>  % +/−  <dig>  %  of the 300 mm values were obtained , excluding the initial 4–20 h timepoints.
acetone  is more selectively analyzed at a wavelength of 265 nm  but in the abe fermentation sample an interfering peak  was present .
by applying a higher wavelength  the area of the interfering peak was greatly reduced .
interestingly, despite the interference, even at 265 nm good analysis data  was obtained.
the results from the 285 nm detection were similar , excluding the 112 h time point .
the increase and decrease of the profiles of ethanol, acetone and n-butanol during the course of the fermentation was caused by an applied membrane pervaporation which removed these components from the fermentation medium.
during the first 50 h, the microbial production outpaced the membrane removal until the accumulated solvents became toxic to the microorganism and stopped further secretion.
at this point, a decline in concentration was observed due to removal by membrane pervaporation .
since the concentration of the solvents was eventually reduced to a sub-toxic level, the cells resumed production of acetone, n-butanol and ethanol later in the experiment .
overall, analysis on the 50 mm guard column was able to monitor these changes with sufficient accuracy and with a sample analysis time of only 5 min compared to about 45 min on the 300 mm column.
application of the bio-rad micro guard cation h guard column
an even shorter guard column is the 30 mm x  <dig>  mm i.d.
bio-rad micro guard cation h .
it can be especially well used for the analysis of compounds that elute late on the 300 mm column.
for example, fig.
9a shows its application to the n-butanol analysis mentioned earlier.
the analysis results were very similar to the ones obtained with the 50 mm guard column with results ranging  <dig> - <dig>  % for dh <dig>   <dig> - <dig>  % for dh1Δacrb and  <dig> - <dig>  % for bw <dig> profile analysis compared to the 300 mm column .
however, by using a standard  <dig>  ml/min flow rate, the analysis time was cut in half  compared to the 50 mm column .fig.
<dig> chromatogram of  the supernatant of escherichia coli strain dh <dig> fermentation showing n-butanol  elution at  <dig>  ml/min flow rate,  the hydrolysate  <dig> showing 5-hmf  and furfural  elution at  <dig>  ml/min flow rate,  a mixture of levulinic acid,  <dig> -hexanedione, 5-hmf,  <dig> -dimethyltetrahydrofuran, furfural, methylfuran, 5-methylfurfural and  <dig> -dimethylfuran at  <dig>  ml/min flow rate,  a standard of n-hexanol  at  <dig>  ml/min flow rate, using the 30 mm guard column.
hplc parameters: column temperature  <dig> °c, mobile phase 5 mm sulfuric acid, flow rate  <dig>  ml/min , refractive index detection
applied to the analysis of 5-hmf and furfural , the analysis time  was further reduced by more than half compared to the 50 mm guard column .
furfural concentrations were slightly higher compared to the 300 mm column:  <dig>  % ,  <dig>  %  and  <dig>  %  for hydrolysate 1– <dig>  respectively .
the concentration for 5-hmf was lower for hydrolysate  <dig>  and hydrolysate  <dig>  but identical for hydrolysate  <dig> .
an interesting application of this guard column is the analysis of some promising liquid fuel molecules and their precursors  <cit> .
most of these types of molecules are usually analyzed by gas chromatography.
if analyzed by liquid chromatography on the 300 mm column, the mobile phase has to be modified with a higher concentration of an organic solvent, e.g.
acetonitrile, in order to reduce their long retention time  <cit> .
instead of changing the mobile phase composition, we have explored the reduction of the retention time by using the 30 mm guard column.
fig.
9c shows the nearly baseline separation of a standard mixture of levulinic acid,  <dig> -hexanedione, 5-hmf,  <dig> -dimethyltetrahydrofuran, furfural, methylfuran, 5-methylfurfural and  <dig> -dimethylfuran, eluting within only 4 min.
since the latter three compounds elute after furfural their elution times are in the range of 1–2 h on the 300 mm column .
interestingly, despite the structural similarity, especially of the furans, the 30 mm guard column provided sufficient separation efficiency for these molecules.
when the flow rate on the 30 mm guard column was increased to 1 ml/min a further reduction of the analysis time was achieved.
at this flow rate, other compounds eluting within the first 25 min on the 300 mm column now eluted in less than 1 min on the 30 mm guard column .
for example, the elution of n-hexanol was at  <dig>  min at a flow rate of 1 ml/min  compared to 96 min on the standard 300 mm column .
in general, the 30 mm guard column operated at  <dig>  ml/min elutes compounds about  <dig> times faster than the 50 mm guard column .
examples are, e.g.,  <dig> -dimethylfuran , n-hexanol  and  <dig> -dimethyltetrahydrofuran .
the 30 mm guard column can therefore be an ideal tool for the rapid analysis of selected late-eluting compounds.
we therefore encourage researchers to explore this guard column for the analysis of compounds that seem to be “invisible” to the rid when injected on the 300 mm column.
this invisibility is most likely due to the effect that compounds remain on the column for a long time and are eluted as broad peaks appearing more like a “baseline-drift”, which makes peak detection and compound quantification difficult or impossible.
CONCLUSIONS
we have used examples from various experiments to show that a 50 mm guard column can be successfully used for the analysis of samples from many bioenergy- and biotechnology-relevant experimental setups.
with this column, samples can be analyzed up to ten-times faster compared to the standard 300 mm column with very comparable results.
therefore, it is an ideal tool for processing a large amount of samples, such as in screening or discovery experiments.
by applying correct peak integration and interference subtraction, concentration profiles from even more complex samples are reliably obtained.
a further decrease of the analysis time was achieved by using a 30 mm guard column that has been shown to be especially suitable for the rapid analysis of compounds with long elution times on the standard 300 mm column, including biofuel-related alcohols  and furan- and tetrahydrofuran-type molecules.
owing to the universal applicability and the ease of use as well the numerous citations in the literature, we suspect that almost all labs involved in the fields of biotechnology or biomass conversion technology are equipped with an hplc-uv/rid instrument and should therefore be able to easily apply the methods presented here.
besides the faster analysis time, guard columns only cost a fraction of their counterparts they were designed to protect.
we therefore encourage researchers to explore the feasibility of applying the separation on a guard column to their experimental setup in order to significantly reduce analysis time.

 a popular liquid chromatography column used for the analysis of biomass conversion and fermentation products is based on a polymer matrix of polystyrene-divinylbenzene. it is operated in isocratic elution mode enabling the connection to a refractive index detector which provides universal compound detection. this set up allows for analysis of a wide range of compounds. with a standard 300 mm length, this type of column provides baseline separation of the main cell wall sugars of many lignocellulosic biomass feedstocks. this column is also used for the analysis of organic acids, alcohols and sugar degradation products. it only requires acidified water as mobile phase and only minimal sample preparation. typical hplc methods aim to separate complex mixtures in order to individually detect isolated compounds – particularly when a non-specific detector like rid is used. in our experience from supporting researchers in an organization comprising more than scientists dedicated to bioenergy research, many analytical questions can be reduced to identifying the change in a few compounds over the course of the experiment. furthermore, reducing analysis time is often more important than obtaining precise absolute quantification data. this is especially true for the results from screening experiments involving a larger amount of samples. the detection of relative changes is often sufficient to identify promising candidates for further analysis. fast methods targeting individual compounds in complex mixtures have previously been established and include, for example, gas chromatography spectroscopy, or enzymatic assays, such as for sugars, sugar alcohols, organic acids, and ethanol. several of these target analytes can even be measured in an automated mode using a biochemistry analyzer and results are obtained in minutes. however, all such methods either require costly equipment or individual analysis kits for every single analyte. therefore, it is tempting to use existing analytical instrumentation and reduce analysis time for higher sample throughput. in this respect, scarlata and hyman have successfully shown that reducing the column length to 100 mm can reduce the analysis time by a factor of five compared to the 300 mm column, but still provide adequate accurate results from a biomass compositional analysis. in our studies, we explored a further reduction of the analysis time by using only a guard column as the simplest and most basic column available. for this purpose, samples generated by various research groups in our institute were analyzed on the standard 300 mm aminex hpx-87h column and compared to results obtained from an analysis on a guard column.  a short column length is often used for faster separation especially in conjunction with mass spectrometric detection. these shorter columns have usually smaller particle sizes which greatly improve resolution. however, columns like the aminex hpx-87h operate in a mixture of size- and ion-exchange/exclusion. they consist of a resin with a rather large particle size. as a result, a reduction in column length significantly lowers column efficiency. for example, a reduction of the column length to 50 mm results in practically unresolved glucose and xylose. these types of columns can therefore not be directly compared to other columns. despite the lower separation efficiency, the shorter columns can still provide adequate chromatographic resolution when used with appropriate samples in which compounds co-eluting with the target analytes are either not present or their concentration is sufficiently low so they do not significantly interfere with quantification. figure  shows the elution profile of some of the most common fermentation products and sugar degradation products, furfural) on the 50 mm rezex rfq fast acid h+ guard column operated at different temperatures at a flow rate of  ml/min. the degradation products 5-hmf and furfural are promising precursors for the generation of biofuels and value-added chemicals. good separation of all these compounds is achieved at a column temperature of °c. at this temperature, ethanol and acetone are almost baseline separated. by increasing the column temperature the alcohols elute later and 5-hmf and furfural elute earlier. the retention time of acetone is practically not affected by temperature. ethanol/acetone separation is deteriorated at a higher temperature and 5-hmf is co-eluting with n-butanol at °c and with iso-butanol at °c. the column temperature must therefore be chosen based on the sample composition so that target analytes do not overlap. for example, as mentioned above, a temperature of °c has to be applied for successful separation of ethanol and acetone. this column temperature is also beneficial for the faster elution of iso-and n-butanol. in contrast, 5-hmf and furfural are eluted faster at a higher temperature. however, depending on which butanol isomer is present, the column temperature needs to be correctly chosen since 5-hmf coelutes either with n-butanol at °c or iso-butanol at °c.fig. separation of ethanol, acetone, iso-butanol, n-butanol, 5-hmf and furfural on the 50 mm guard column at a column temperature of °c, °c and °c.hplc parameters: 50 mm ×  mm rfq fast acid h+ column, mobile phase 5 mm sulfuric acid, flow rate  ml/min, refractive index detection using a 20 μl injection volume, the determined linear calibrations for the compounds under study ranged from -  mg/ml to 50 mg/ml with ri and uv detection. uv detection resulted in an about 100-fold increased sensitivity for 5-hmf and furfural compared to ri detection.table calibration results for each compound chromatography conditions: 50 mm ×  mm rfq fast acid h+ kept at °c, flow rate:  ml/min of 5 mm sulfuric acid, 20 μl injection volume; lower limit of the calibration range was determined as the concentration level with a relative standard deviation <10 % since °c is close to the maximum operating temperature of the column, we chose a column temperature of °c for most of the following experiments. this is good balance between fast analysis time and column life time. application to analysis of n-butanol and iso-butanol production during fermentations these two alcohols elute later than most other components present in the fermentation media. fig. 2a shows a representative chromatogram from an e.coli fermentation producing n-butanol which eluted at  min. fig. 2b shows the determined n-butanol production of three e. coli strains after incubation for 24 h, 48 h and 72 h. the n-butanol profile obtained with the 50 mm guard column was very similar compared to the 300 mm bio-rad hpx-87h column. values reached -  % of the values obtained with the longer column. the dh strain is able to produce more n-butanol than bw which almost showed no significant n-butanol levels. usually, bw produces about half the concentration of dh. unexpectedly, the bw strain used in this experiment did not perform as expected. the deletion of acrb efflux pumps in dh1Δacrb lead to even higher n-butanol levels. acrb pumps are large proteins and are used to actively secret n-butanol from the cell. however, they can be considered “energy drainers” since they use a big portion of the cellular resources needed for n-butanol synthesis. at the levels measured here, n-butanol is not toxic for the e. coli cells and therefore the acrb pumps present in the dh strain are not a “de-toxification”-advantage over the dh1Δacrb strain which has more energy resources available for n-butanol production.fig. representative chromatogram of the supernatant from escherichia coli strain dh fermentation analyzed with the 50 mm guard column showing n-butanol elution, and n-butanol concentration of the supernatant from e. coli strains dh dh1Δacrb and bw fermentations after 24 h, 48 h and 72 h analyzed with the 50 mm guard column, 300 mm column and 30 mm guard column. hplc parameters: column temperature 55 °c, mobile phase 5 mm sulfuric acid, flow rate  ml/min, refractive index detection a similar chromatogram was obtained for iso-butanol production from e. coli strains sfab and sfab. iso-butanol eluted at  min in a region where no interference from other media components were expected. the values measured with the 50 mm guard column were again almost identical to the ones obtained with the 300 mm column. in this experiment, the decrease of iso-butanol after 48 h for the sfab strain was explained by evaporation due to a leak in the cap of the flask.fig. representative chromatogram of the supernatant from escherichia coli strain sfab fermentation analyzed with the 50 mm guard column showing iso-butanol elution, and iso-butanol concentration profile of the supernatant from e. coli strains sfab and sfab fermentation between 24 h and 192 h analyzed with the 50 mm guard column and 300 mm column. hplc parameters: column temperature 55 °c, mobile phase 5 mm sulfuric acid, flow rate  ml/min, refractive index detection the analysis of n- and iso-butanol was performed with a 4 min total run time and this is about 9– times faster compared to the standard 300 mm column. application to analysis of 5-hydroxymethylfurfural and furfural 5-hmf and especially furfural are compounds with long elution times on the standard 300 mm hpx-87h column. experiments requiring the analysis of these compounds often include determination of the degree of their removal or reduction during membrane detoxification of hydrolysate or the rate of sugar degradation during pretreatment or conversion steps. for the purpose of this study, three biomass hydrolysates prepared under different severity conditions were analyzed. fig. 4a depicts the elution profiles for the two compounds on the 50 mm guard column. hydrolysate was prepared with a lower sulfuric acid concentration and incubated at a lower temperature causing less sugar degradation. therefore, it shows a lower 5-hmf and a lower furfural concentration compared to the other two hydrolysates which were prepared with a higher acid concentration and a higher temperature. as illustrated in fig. 4b, the 5-hmf analysis results with the 50 mm guard column compared to the 300 mm column were lower for hydrolysate, lower for hydrolysate and lower for hydrolysate. however, the furfural amounts determined with the 50 mm guard column were in the range of  %-  % of the 300 mm column results and can be considered very comparable. in conclusion, the different pretreatment severity profiles were adequately reflected by analysis with the 50 mm guard column.fig. chromatogram of hydrolysate hydrolysate and hydrolysate analyzed with the 50 mm guard column showing 5-hmf and furfural elution, and 5-hmf and furfural concentrations of hydrolysate hydrolysate and hydrolysate analyzed with the 50 mm guard column, 300 mm column and 30 mm guard column. hplc parameters: column temperature °c, mobile phase 5 mm sulfuric acid, flow rate  ml/min, refractive index detection application to analysis of ethanol production and xylose and glucose consumption during fermentations when glucose and xylose are present in a mixture, their accurate determination using the 50 mm guard column is challenging since the separation of these two sugars is practically not possible. however, the analysis can be performed when either one of the two is present in high abundance or one of the two monosaccharides concentrations is constant over the course of the experiment. ethanol eluted at  min which was close to other fermentation media compounds but was still sufficiently isolated for selective measurement in these experiments.fig. representative chromatogram of the supernatant from a saccharomyces cerevisiae sa-1-x strain fermentation analyzed with the 50 mm guard column showing xylose and ethanol elution, and xylose and ethanol concentration profile of the supernatant from a modified s. cerevisiae strain fermentation between 0 h and 62 h analyzed with the 50 mm guard column and 300 mm column. hplc parameters: column temperature 55 °c, mobile phase 5 mm sulfuric acid, flow rate  ml/min, refractive index detection fig. chromatogram of the supernatant from saccharomyces cerevisiae sa- strain fermentation supplemented with 25 % of ph adjusted hydrolysate analyzed with the 50 mm guard column showing glucose, xylose and ethanol elution after 42 h, 50 h and 67 h, and xylose and ethanol concentration profile of the supernatant between 0 h and 62 h from a s. cerevisiae sa- strain fermentation supplemented with 25 % of pervaporation detoxified hydrolysate and 25 % of ph adjusted hydrolysate analyzed with the 50 mm guard column and 300 mm column. hplc parameters: column temperature °c, mobile phase 5 mm sulfuric acid, flow rate  ml/min, refractive index detection the consumption of xylose and production of ethanol of a modified saccharomyces cerevisiae strain analyzed with the 50 mm guard column and 300 mm column is shown in fig. 5b and a representative chromatogram is shown in fig. 5a. the xylose amounts measured with the 50 mm guard column were in the range of -  % and the ethanol amounts were in the range of -  % of the values obtained with the 300 mm column. the two lower concentrations of ethanol showed  % and  % deviation most likely due to interfering co-eluting compounds at these lower concentrations. overall, the consumption/production profile measured with the 50 mm guard column is very similar to the one obtained with the 300 mm column. however, the total run time using the 50 mm guard column was only 3 min and ethanol elution was completed at  min. this is about times faster compared to the 300 mm column. in another experiment, s. cerevisiae sa- was grown in a medium supplemented with 25 % hydrolysate that was either ph adjusted or detoxified by membrane pervaporation which reduced the concentration of toxic compounds such as furfural, formic and acetic acid. the hydrolysate was obtained by a dilute acid and heat pretreatment of miscanthus biomass in order to release hemicellulosic sugars and improve cellulose digestibility. during this process, the harsh conditions lead to sugar and lignin degradation resulting in the production of a fleet of fermentation inhibitors like furans, phenolics and organic acids which could also interfere with the analysis. representative chromatograms of the sample with ph-adjusted hydrolysate at time points 42 h, 50 h and 67 h analyzed with the 50 mm guard column are shown in fig. 6a-c. a decrease of glucose and increase of ethanol can be observed over the time course whereas the remaining chromatogram is mostly the same. the inset in fig. 6a shows the close elution of glucose and xylose resulting in a peak with a shoulder making accurate integration difficult. fig. 6d and 6e show the measured glucose and ethanol concentrations over the course of the experiments with the membrane-detoxified hydrolysate and the ph-adjusted hydrolysate, respectively. the values for “glucose uncorrected” were obtained when the attempt was made to integrate glucose separately from the closely-eluting xylose peak. the determined “glucose uncorrected” concentrations for experiments with the ph-adjusted and with the detoxified hydrolysate were -  % and -  %, respectively, of the concentrations obtained when analyzed with the 300 mm column. this indicates that xylose interfered with a correct glucose area determination. the ethanol concentrations were almost identical with -  % and -  %, respectively, compared to the results of the 300 mm column. when glucose and xylose were integrated together as one peak and the area of the xylose peak of the 67 h time point was subtracted, “glucose corrected” values were obtained which were closer to the results of the 300 mm column. even without glucose correction, the effect of the toxic hydrolysate on delaying the glucose fermentation and production of ethanol until around 42 h into the fermentation was clearly observed with the 50 mm column. application to analysis of glucose release from enzymatic digestibility assays of pretreated biomass the enzymatic release of glucose from cellulose after biomass pretreatment is often performed not only to determine of the effectiveness of the pretreatment itself but also for the evaluation of the hydrolytic potential of new enzymes. a commonly used method is the pretreatment of biomass with dilute acid at higher temperatures which leaves behind a solid cake consisting of mainly lignin and decrystallized cellulose. after washing and/or neutralization this cake is incubated with cellulose-degrading enzymes in either citrate or acetate buffer at ph  in order to release glucose. as shown in the chromatogram of the supernatant after enzymatic digestion of “pretreated miscanthus biomass”, citrate/citric acid of the citrate buffer is interfering with glucose at lower and higher concentrations on the 50 mm guard column. furthermore, sorbitol derived from the commercial enzyme mixture also interferes, but to a much lower extent. using the 300 mm column, citric acid, glucose and sorbitol can be separated. as observed earlier with the glucose/xylose integration, the attempt to integrate the glucose peak separately lead to glucan conversions that deviated by -  % from the ones obtained using the 300 mm column. for example, glucan conversion after 72 h was  % vs  %. at time point 0 h, the observed glucose peak was derived from the enzyme mixture used in the experiment. therefore, this area had to be subtracted as a blank from the glucose area of following time points. similar as before, if the citric acid and glucose peaks at timepoint 0 h were integrated as one peak and subtracted from following peaks the values obtained with the 50 mm guard column were in the range of -  % of the values with the 300 mm column. the glucan conversion after 72 h was  % vs  %. the elution on the 50 mm guard column is completed after  min and is therefore about times faster compared to  min with the 300 mm column.fig. chromatogram of the supernatant from the enzymatic digestion of “pretreated miscanthus biomass” showing citric acid, glucose, sorbitol and acetic acid elution after 0 h and 24 h digestion in citrate buffer analyzed with the 50 mm guard column, 0 h and 24 h digestion in citrate buffer analyzed with the 300 mm column, 24 h digestion in acetate buffer analyzed with the 50 mm guard column; and enzymatically released glucan concentration profile of the supernatant from the enzymatic digestion of “pretreated miscanthus biomass”between 0 h and 72 h using citrate buffer acetate buffer analyzed with the 50 mm guard column and 300 mm column. hplc parameters: column temperature °c, mobile phase 5 mm sulfuric acid, flow rate  ml/min, refractive index detection. error bars represent +/− standard deviation using acetate buffer, only sorbitol derived from the commercial enzyme mixture interfered with glucose on the 50 mm guard column because acetate eluted later at  min. a “split-peak” integration of glucose and sorbitol is sufficient to obtain glucan conversions within -  % of the ones resulting from using the 300 mm column. the 72 h conversion was  % vs  % and the run time was again about times faster. it is noteworthy to mention that the acetate buffered samples can also be run with an analysis time of only  min as the citrate buffered samples. the acetate peak will then be eluted within the first  min of the next sample. furthermore, the sample analysis time of  min is fast enough to compete with other higher-throughput glucose methods like using a ysi bioanalyzer for glucose determination. with the “overlap injection” function of the autosampler, which allows rapid injection of the next sample after a run is completed, about samples can be processed per day. application to analysis of ethanol, acetone and n-butanol production during acetone-butanol-ethanol fermentation the analysis of the products from acetone-butanol-ethanol fermentation with a short column is challenging since clostridia secret a fleet of other compounds which can co-elute with the target analytes. the use of an additional uv detector is beneficial for more accurate compound detection and quantification. fig. 8a shows the chromatogram of a representative sample with ri detection. ethanol and n-butanol have to be analyzed using the unspecific ri detector. for n-butanol, the concentration profile obtained with the 50 mm guard column is very similar to the 300 mm column, since interfering compounds were absent or in low concentration at this retention time. the measured n-butanol concentrations were on average  % +/−  % of the values obtained with the 300 mm column, except for early time points due to lower concentrations affecting accuracy, and the 90 h and 112 h time points with  % and  % deviation.fig. chromatogram of the supernatant from a clostridium acetobutylicum abe fermentation after 23 h analyzed with the 50 mm guard column showing ethanol, n-butanol, ethanol interference, acetone interference and acetone analyzed with refractive index detection uv 210 nm detection uv 265 nm detection, uv 285 nm detection, and concentration profile of the supernatant from a chlostridium acetobutylicum abe fermentation of n-butanol, acetone, ethanol, between 0 h and 112 h analyzed with the 50 mm guard column and 300 mm column. hplc parameters: column temperature °c, mobile phase 5 mm sulfuric acid, flow rate  ml/min, refractive index and uv detection in the 210 nm uv trace of the same sample a compound is visible that is interfering with the ethanol determination using ri detection. however, with the 300 mm column, peak and ethanol can be separated. if ethanol was determined only by ri detection, concentrations were overestimated and the values ranged -  % of the values obtained with the 300 mm column. the interfering compound was present early in the fermentation process when ethanol production was zero or very minimal. therefore, it should be possible to measure the area of peak with 210 nm uv detection and correlate it to the area obtained with ri detection. in this way, the interference of peak in rid mode should be reduced by measuring the sum of the area with rid and subtracting the theoretical “rid area contribution” of peak calculated from its 210 nm uv area. however, we noticed that the low intensity of peak in the earlier time points made correlation of uv and rid signal less accurate. we therefore used an empirical method to determine the uv/rid correlation by adjusting the correlation factor so the corrected ethanol concentration from the 50 mm guard column best matched the 300 mm column results. this correction factor was applied to the uv-to-rid conversion of peak at all time-points of the fermentation. “ethanol corrected” values of average  % +/−  % of the 300 mm values were obtained, excluding the initial 4–20 h timepoints. acetone is more selectively analyzed at a wavelength of 265 nm but in the abe fermentation sample an interfering peak was present. by applying a higher wavelength the area of the interfering peak was greatly reduced. interestingly, despite the interference, even at 265 nm good analysis data was obtained. the results from the 285 nm detection were similar, excluding the 112 h time point. the increase and decrease of the profiles of ethanol, acetone and n-butanol during the course of the fermentation was caused by an applied membrane pervaporation which removed these components from the fermentation medium. during the first 50 h, the microbial production outpaced the membrane removal until the accumulated solvents became toxic to the microorganism and stopped further secretion. at this point, a decline in concentration was observed due to removal by membrane pervaporation. since the concentration of the solvents was eventually reduced to a sub-toxic level, the cells resumed production of acetone, n-butanol and ethanol later in the experiment. overall, analysis on the 50 mm guard column was able to monitor these changes with sufficient accuracy and with a sample analysis time of only 5 min compared to about 45 min on the 300 mm column. application of the bio-rad micro guard cation h guard column an even shorter guard column is the 30 mm x  mm i.d. bio-rad micro guard cation h. it can be especially well used for the analysis of compounds that elute late on the 300 mm column. for example, fig. 9a shows its application to the n-butanol analysis mentioned earlier. the analysis results were very similar to the ones obtained with the 50 mm guard column with results ranging -  % for dh -  % for dh1Δacrb and -  % for bw profile analysis compared to the 300 mm column. however, by using a standard  ml/min flow rate, the analysis time was cut in half compared to the 50 mm column.fig. chromatogram of the supernatant of escherichia coli strain dh fermentation showing n-butanol elution at  ml/min flow rate, the hydrolysate showing 5-hmf and furfural elution at  ml/min flow rate, a mixture of levulinic acid, -hexanedione, 5-hmf, -dimethyltetrahydrofuran, furfural, methylfuran, 5-methylfurfural and -dimethylfuran at  ml/min flow rate, a standard of n-hexanol at  ml/min flow rate, using the 30 mm guard column. hplc parameters: column temperature °c, mobile phase 5 mm sulfuric acid, flow rate  ml/min, refractive index detection applied to the analysis of 5-hmf and furfural, the analysis time was further reduced by more than half compared to the 50 mm guard column. furfural concentrations were slightly higher compared to the 300 mm column:  %,  % and  % for hydrolysate 1– respectively. the concentration for 5-hmf was lower for hydrolysate and hydrolysate but identical for hydrolysate. an interesting application of this guard column is the analysis of some promising liquid fuel molecules and their precursors. most of these types of molecules are usually analyzed by gas chromatography. if analyzed by liquid chromatography on the 300 mm column, the mobile phase has to be modified with a higher concentration of an organic solvent, e.g. acetonitrile, in order to reduce their long retention time. instead of changing the mobile phase composition, we have explored the reduction of the retention time by using the 30 mm guard column. fig. 9c shows the nearly baseline separation of a standard mixture of levulinic acid, -hexanedione, 5-hmf, -dimethyltetrahydrofuran, furfural, methylfuran, 5-methylfurfural and -dimethylfuran, eluting within only 4 min. since the latter three compounds elute after furfural their elution times are in the range of 1–2 h on the 300 mm column. interestingly, despite the structural similarity, especially of the furans, the 30 mm guard column provided sufficient separation efficiency for these molecules. when the flow rate on the 30 mm guard column was increased to 1 ml/min a further reduction of the analysis time was achieved. at this flow rate, other compounds eluting within the first 25 min on the 300 mm column now eluted in less than 1 min on the 30 mm guard column. for example, the elution of n-hexanol was at  min at a flow rate of 1 ml/min compared to 96 min on the standard 300 mm column. in general, the 30 mm guard column operated at  ml/min elutes compounds about times faster than the 50 mm guard column. examples are, e.g., -dimethylfuran, n-hexanol and -dimethyltetrahydrofuran. the 30 mm guard column can therefore be an ideal tool for the rapid analysis of selected late-eluting compounds. we therefore encourage researchers to explore this guard column for the analysis of compounds that seem to be “invisible” to the rid when injected on the 300 mm column. this invisibility is most likely due to the effect that compounds remain on the column for a long time and are eluted as broad peaks appearing more like a “baseline-drift”, which makes peak detection and compound quantification difficult or impossible.  we have used examples from various experiments to show that a 50 mm guard column can be successfully used for the analysis of samples from many bioenergy- and biotechnology-relevant experimental setups. with this column, samples can be analyzed up to ten-times faster compared to the standard 300 mm column with very comparable results. therefore, it is an ideal tool for processing a large amount of samples, such as in screening or discovery experiments. by applying correct peak integration and interference subtraction, concentration profiles from even more complex samples are reliably obtained. a further decrease of the analysis time was achieved by using a 30 mm guard column that has been shown to be especially suitable for the rapid analysis of compounds with long elution times on the standard 300 mm column, including biofuel-related alcohols and furan- and tetrahydrofuran-type molecules. owing to the universal applicability and the ease of use as well the numerous citations in the literature, we suspect that almost all labs involved in the fields of biotechnology or biomass conversion technology are equipped with an hplc-uv/rid instrument and should therefore be able to easily apply the methods presented here. besides the faster analysis time, guard columns only cost a fraction of their counterparts they were designed to protect. we therefore encourage researchers to explore the feasibility of applying the separation on a guard column to their experimental setup in order to significantly reduce analysis time. 
results from the guard columns were compared to the analyses using the standard 300 mm aminex hpx-87h column which is widely applied to the analysis of samples from many biotechnology- and bioenergy-related experiments such as biomass conversions or fermentations. if correct peak integration and interference subtraction was applied, concentration profiles from enzymatic digestibility experiments and even more complex samples fermentation) were reliably obtained. in chromatography, one strategy is to reduce column length for decreased analysis time. a further decrease in analysis time was achieved by using the 30 mm micro guard cation h guard column. therefore, the feasibility of analyzing samples simply on a guard column was explored using refractive index and ultraviolet detection. compound profiles in fermentation samples were analyzed with similar accuracy compared to results using the 300 mm column. with the 50 mm guard column, samples were analyzed up to ten-times faster compared to the 300 mm column. this column is especially suitable for the rapid analysis of compounds with long elution times on the standard 300 mm column, such as biofuel-related alcohols and furan- and tetrahydrofuran-type molecules. the 50 mm rezex rfq fast acid h+ guard column was able to separate the most common fermentation products and promising precursors of biofuels and value-added chemicals. therefore, it is an inexpensive and ideal tool for processing a large amount of samples, such as in screening or discovery experiments, where detecting relative changes is often sufficient to identify promising candidates for further analysis. applied to a suitable set of samples, separations on a guard column can give rapid and sufficiently accurate information on compound changes over the course of an experiment. increasing sample throughput is needed when large numbers of samples have to be processed.
 increasing sample throughput is needed when large numbers of samples have to be processed. in chromatography, one strategy is to reduce column length for decreased analysis time. therefore, the feasibility of analyzing samples simply on a guard column was explored using refractive index and ultraviolet detection. results from the guard columns were compared to the analyses using the standard 300 mm aminex hpx-87h column which is widely applied to the analysis of samples from many biotechnology- and bioenergy-related experiments such as biomass conversions or fermentations.  the 50 mm rezex rfq fast acid h+ guard column was able to separate the most common fermentation products and promising precursors of biofuels and value-added chemicals. compound profiles in fermentation samples were analyzed with similar accuracy compared to results using the 300 mm column. however, separation of glucose and xylose was not achieved. nevertheless, it was possible to monitor the consumption of one of the two sugars during fermentation if the other one was absent or remained constant over the course of the experiment. if correct peak integration and interference subtraction was applied, concentration profiles from enzymatic digestibility experiments and even more complex samples fermentation) were reliably obtained. with the 50 mm guard column, samples were analyzed up to ten-times faster compared to the 300 mm column. a further decrease in analysis time was achieved by using the 30 mm micro guard cation h guard column. this column is especially suitable for the rapid analysis of compounds with long elution times on the standard 300 mm column, such as biofuel-related alcohols and furan- and tetrahydrofuran-type molecules.  applied to a suitable set of samples, separations on a guard column can give rapid and sufficiently accurate information on compound changes over the course of an experiment. therefore, it is an inexpensive and ideal tool for processing a large amount of samples, such as in screening or discovery experiments, where detecting relative changes is often sufficient to identify promising candidates for further analysis. keywords ion exclusion chromatographyaminex hpx-87hrezex rfqmicro guard cation henzymatic digestibility assayglucoseethanolbutanolfuransfermentationissue-copyright-statement© the author 2015 