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Food Ingredient Structure and Function ONPS2549 and ONPS2553 Report on carbohydrate analysis by HPLC Instructions Use this template to: 1. Record any relevant information about your sample(s) during the practical session using the form on page 2 of this handout; 2. Calculate your data using the second form (currently pages 3-9 of this document) by working your way systematically through each stage, recording each step, and including units for all data (other than the peak areas or height values from your chromatographic data, which have arbitrary units); 3. Transfer the final results from the calculation to your report which should be written here; 4. When completed, this report will have two appendices (one is the form for anything you recorded during the lab session and the second is the calculation form; 5. Do not include any lab results or raw data as part of this main body of your report, other than that which is specifically needed or requested; 6. You should add or delete page breaks in this document, to suit your requirements; 7. Delete these instructions before lodging your report; and 8. Save and then lodge this Word file on-line directly to the folder of the demonstrator who worked with you in the lab session.   Food Ingredient Structure and Function ONPS2549 and ONPS2553 Recording form for carbohydrate analysis by HPLC (becomes Appendix A) This form is part of your final report. Remember to include units when you record your results and in the calculations wherever appropriate. Your name: Student number: Group: Partner(s) for this exercise: Name of your food product: If you prepared your own solution of your food ingredient/product then record here any relevant information that may be needed for your calculations. This would include the exact weight of sample taken for analysis. Note that for this analysis, there may be no label data to record during your lab session, however, there is a note in the module Lab manuals: “Label and spec sheet data for glucose analysis labs” This resource sheet provides information that will help you when you write your report and evaluate your results if you have analysed glucose syrup or a maltodextrin by HPLC:   Food Ingredient Structure and Function ONPS2549 and ONPS2553 Form for calculating results for carbohydrate analysis by HPLC (becomes Appendix B) Scope This handout is a form designed to assist you in calculating your HPLC results. It covers the interpretation of the chromatograms as well as calculating the analysis results for your product. Once completed, this should be included in your report as an appendix. Instructions • Use this form by answering the questions and entering details for each step in the calculation, using the spaces provided; • Include units at each stage in the calculations (wherever appropriate) and particularly with your final results; • Remember to avoid rounding at the intermediate stages in the calculation as this can introduce “rounding errors”; • Unless you have replicate analyses for your sample it will not be possible to estimate the precision. Despite this, it is still really important to round your final result appropriately and this issue is considered at step 13 of this handout. • When completed, retain these sheets as part of your final report, as an appendix. Calculation step 1 Consider your raw data Your lab data probably includes print-outs of chromatograms obtained for the solution prepared using your food along with those for one or more standard sugars. The other information that you will need is in the manual or which you may have recorded, describing the details on preparing solutions for standards and your specific food product or ingredient. Briefly, the background you need to remember is: • The printouts include the graph which is referred to as the chromatogram (the instrument is known as a chromatograph). This has elution time or volume on the horizontal axis and detector response on the vertical axis. Remember that the units for the detector response are not important to us, as long as we are applying the “Same, same, same rule” and running standards and samples at the same time and under the same conditions. This allows us to directly compare the values, so units are not relevant. • We can identify a particular peak based upon the time it eluted from the column. This is also referred to as the retention time and usually the units for this are minutes. • There may be peaks in the chromatograms that you cannot readily identify, and this applies both for standards as well as the sample of your food. It is quite normal for us to be unsure about some of the peaks. • Once we have worked out which peak in our sample corresponds to the glucose and/or sucrose standards, it is possible to calculate how much is present. For this quantification, we can use either the peak height or the peak area values. These are conveniently provided from the computer output accompanying the chromatogram. • Using peak heights or peak areas will usually give us the same results for our calculations, so the choice of which we should use is up to us. • The table prepared by the computer lists the area and height (both in arbitrary units). In addition, there is a column headed Conc (short for concentration). Note carefully that these concentrations are not the amount of a sugar in the solution that we analysed. • The conc values can be useful sometimes but you need to understand how they are calculated along with their limitations or you may encounter problems. The Conc values for each peak are the proportion the peak area represents of the total area measured for all of the peaks recognised by the computer program. The proportions are expressed as percentage values. We can encounter problems if we use these unless we know what molecular component each peak corresponds to and whether all of them are actually carbohydrates. • It may also be helpful for you to understand that we have long experience with this HPLC system and this particular one separates primarily on the basis of molecular size. So monosaccharides will elute first, followed by disaccharides, DP 3,then DP 4 and so on. However, it does not readily help us with really large carbohydrate chains. So the largest size we will normally see is still an oligosaccharide with DP = 8 or 9. Longer molecules may be present in a sample but the system does not separate or show them satisfactorily. Calculation step 2 Select appropriate units for your final answer Your aim in calculating is to ultimately express the amount of glucose and/or sucrose in the food product or ingredient you have analysed. So you need to decide on suitable units for the final results. Do this by considering what units have been used for the data that you plan on comparing your own results with (for example, label information, composition databases or specification sheets). We can only fairly compare numbers which are expressed in the same units. Units chosen: Reason for selection:   Calculation step 3 Draw a simple flow diagram of the analysis procedure This can be simple and outlines the procedure you used in the laboratory. This forms the basis of the remainder of the calculation. You now work your way systematically through each step in the diagram, starting from the last point in the sequence, up to the initial step in the procedure. Note: if any modification was made to the procedure described in the manual then these steps may need to be added to the diagram as they probably affect the calculation of results. An example would include any additional dilution step if you sample was initially too concentrated for analysis. Sub-sample of food product weighed accurately as described in the manual  Sub-sample dissolved into solution and made up to volume specified for the particular food product  Pass through 0.45µm filter  Analyse by injecting into HPLC instrument Calculation step 4 Write and balance any relevant chemical equation This step is required for many chemical/biochemical analyses as the calculation depends upon the stoichiometry of the reaction. However, for this HPLC analysis, no reactions are involved and, therefore we do not need to consider equations any further. Calculation step 5 Identification of peaks from chromatograms of standards Based on the information given at step 1, we use retention time for identification. 1. Firstly, consider the chromatogram for the glucose standard. How many peaks has the computer actually recognised? Is there one peak which is much bigger than any others present? If there is then we can assume that that corresponds to glucose. Then we can enter the retention time value for that into the table below this box. 2. In the glucose chromatogram you may notice a very small peak eluting before the glucose molecules. This is often observed in the chromatographic system (column/ liquid/instrument/detector combination) you used and it does not correspond with any standard we have ever tried. Therefore it is perfectly valid to ignore it completely. 3. Another interesting issue with the chromatogram of the glucose standard, is that there may be another small peak eluting after that of glucose. If it occurred in your chromatogram, you might like to think about what this represents and why it is there. Usually the amount is so small that it will have relatively little influence on the calculated results or the reliability of the analysis. 4. If you also ran a sucrose standard, then the chromatogram can be evaluated in a similar way. Again, enter the elution time for sucrose in the table below. You might like to consider what your chromatogram tells you about the purity of the sucrose used to prepare the solution used as the standard. Table of results for standard sugars1 Sugar Elution time (min) Peak area2 Glucose Maltose Note: 1 Additional rows could be added if we were able to run additional standards 2 This is taken as the time corresponding to the highest point of the curve for the peak and the units are not important Calculation step 6 Interpretation of chromatograms for samples Now that we know the time at which the known standards eluted, if we find a peak in our food sample that has eluted at the same time then we can be reasonably sure that it is the same sugar as that in the chromatogram of the standard sugar. So, is there a peak in the chromatogram for your food sample corresponding to glucose, and one for maltose? Note here that retention times may vary slightly, particularly if the reading is taken to two or three decimal places. Glucose present: (Yes/No/delete one) Maltose present: (Yes/No/delete one) How many other peaks do you think are present in the chromatogram of your food sample? Calculation step 7 Consider your calibration Often for chromatographic analyses we would run a series of standards having a range of concentrations and then we would draw a calibration curve, but for this HPLC analysis we can actually simplify the calibration step. The approach is referred to as a single point calibration. So, although we could use Excel and draw a graph, it is not actually necessary. Instead we can use the following equation which is another way of describing a straight line relationship between absorbance and the amount of glucose: Concentration of glucose (food solution) = Peak area (food solution) Concentration of glucose (standard) Peak area (standard) This equation works as long as the peak areas were obtained on the standard and sample in accordance with the “Same, same, same rule”. In this equation, what we want to calculate is the item shown in bold italics. So we can rearrange the equation and note also that the units for our concentration of glucose in our sample will be the same as the units we selected or used for concentration of the glucose standard. From the manual, you prepared the standard solution with a concentration of 0.5%, which is another way of saying 0.5g per 100mL. If you made it up to be different from that then you should use your own value. So now calculate the glucose concentration for your sample: Calculation step 8 Calculating how much glucose was in the original food • Step 7 has given us the amount of free glucose in 100mL of the solution; • It may help you to look back at the flow diagram at step 3 above; • Ask yourself where all the free glucose in the 100mL came from. This was the weight of the food sample we took. So now we know how much glucose there was in that weight of food. So enter this information here: (for example: 0.0956g glucose in 0.5g of malt) Calculation step 9 How much glucose would there have been in 1.000g of the sample? Calculation step 10 Now express this value in terms of the units that you selected to use at step 2 on page two? Calculation step 11 Review your units Check that your units are appropriate. Do we need to convert mg to g of glucose this time? Calculation step 12 Precision and rounding Precision values can only be readily estimated if we have replicate values (for example, two peak area measurements for glucose in our food sample). If we cannot estimate precision, it is still very important to round because the number of significant figures you present in your final calculated value communicates the level of confidence you have in the result. So the rule used here is to consider any measurements that were used in the calculation of your results. Then we will decide which of these has the least number of significant figures. Then we will express our result to the same number of figures. So for example, if we weighed a sample using a four decimal place balance (weight of 0.5014g) and a volumetric flask (100mL) that would be a very accurate value (probably 100.2mL) and the syringe system in the HPLC to inject the 20µL volumes is also quite reliable (probably 20.0 ± 0.3µL). In this case the least number of significant figures would be three, associated with the injector syringe. Therefore we would express our final calculated result to no more than that number of significant figures. Can you think of any other number that was relevant to your calculation and which should be considered here? Calculation step 13 Present final result for free glucose Present this in a suitable form, rounded appropriately: Calculation step 14 Calculating content of any other sugar (if present) If you identified a peak for maltose in the chromatogram for your own food sample (Step 6) then now calculate the content of that sugar using the same approach as used for glucose (Steps 7-13). Remember to transfer your answer(s) and present in your report, preferably as part of a table of results.

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• Aim
The aim of this practice was to establish a method for carbohydrate quantification by HPLC, to run standard samples and use the method to determine carbohydrate content in maltodextrin and glucose syrup samples.

• Methods
The method for the analysis of carbohydrates by HPLC is described in laboratory manual [1]. It was followed without modifications. The samples used were commercial maltodextrin preparations with comertial names Fieldose (F30) and Fieldose (F17), manufactured by Starch Australia, with different dextrose equivalents, and glucose syrup with brand name Queen. Glucose and maltose were used as standards. The samples and standards solutions were prepared by dissolving commercial preparations as indicated in the manual and in appendix A table below (both samples and standards).

• Results
The result of maltodextrin and glucose syrup analysis of carbohydrate content by HPLC is given in the following table....

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