Food Analysis with Shodex columns. Saccharides, Organic acids, Vitamins, Fatty acids and Amino acids TECHNICAL NOTEBOOK. No.3

Transcription

1 Food Analysis with Shodex columns Saccharides, Organic acids, Vitamins, Fatty acids and Amino acids TECHNICAL NOTEBOOK No.

2 Contents. Introduction. Saccharides. Separation Mechanism. Monosaccharides and Disaccharides. Saccharides and Sugar Alcohols. WaterSoluble Low Molecular Weight Dietary Fibers. Saccharides with Amino Acids or Organic Acids. Polysaccharides. Sweeteners. Organic Acids. Separation Mechanism. Organic Acids. Vitamins. Fatsoluble Vitamins. Watersoluble Vitamins. Other Ingredients in Food. Higher Fatty Acids. Nucleic Acids as Taste Components. Amino Acids. Appendix A. List of elution volume of saccharides (all columns) B. List of elution volume of saccharides and organic acids (concentration of eluent, SH) C. List of elution volume of organic acids (concentration of eluent, KC8) D. List of elution volume of organic acids (column temperature, KC8)

3 . Introduction In recent years, a broad range of analyses, including agrochemical residues and food additives, have been performed on foods with increasingly greater concern about their safety. This article describes analyses of general food ingredients, especially those based on high performance liquid chromatography (HPLC).. Saccharides HPLCbased analyses of saccharides and sugar alcohols are performed in various modes, such as normal phase, ligand exchange, size exclusion, and ion exchange. There are two series of Shodex columns for sacchride analysis: those operated mainly in ligand exchange mode (SUGAR series), and those in normal phase mode (Asahipak NHP polymeric amino columns). In the SUGAR series columns, a hard styrenedivinylbenzene copolymer designed for saccharide analysis is used as the base material, and a strong cation exchange resin incorporating functional sulfogroups coupled with metal counter ions is used as the packing. Columns of the SUGAR series are available in types according to counter ion, pore size, and pore structure, showing differential selectivity for different sacchrides. On the other hand, the Asahipak NHP columns comprise a hydrophilic polymer gel [poly(vinyl)alcohol] and a polyamine that is chemically and stably bound to the gel. As such, these columns have excellent durability without the problem of timerelated deterioration in silicabased amino columns. Both these series are suitable for saccharide separation and analysis of foods, areas of biochemistry, and natural substances. Other items include the Asahipak GS HQ polymeric packed columns for size exclusion mode to analyze watersoluble, low molecular weight dietary fibers, and the OHpak SB8 HQ polymeric packed columns for size exclusion mode to analyze polysaccharides, as well as the SUGAR KS 8 columns. Table shows the lineup of Shodex packed columns for saccharide analysis. Table. Columns for saccharide analysis () Product Code F8 F8 F8 F8 F8 F9 F8 F8 F F F F F8 F8 F8 F8 F8 F8 F F8 F F F9 F F F F F F F9 Product Name SUGAR SH SUGAR SH8 SUGAR SHG SUGAR SC SUGAR SC8 SUGAR SCLG SUGAR SP8 SUGAR SPG SUGAR SC SUGAR SCG SUGAR SZ SUGAR SZG SUGAR KS8 SUGAR KS8 SUGAR KS8 SUGAR KS8 SUGAR KS8 SUGAR KS8 SUGAR KSG SUGAR KS8 SUGAR KS8G Asahipak GS HQ Asahipak GSG B Asahipak NHP D Asahipak NHP E Asahipak NHPG A Asahipak NHP D Asahipak NHPG A Asahipak NHPLF USPpak MN Counter Ion H + H + H + Ca + Ca + Ca + Pb + Pb + Ca + Ca + Zn + Zn + Na + Na + Na + Na + Na + Na + Na + Na + Na + Ca + Separation Mode SEC + IEX SEC + IEX SEC + LEX SEC + LEX SEC + LEX NP + LEX NP + LEX SEC + LEX SEC + LEX SEC SEC SEC SEC SEC SEC NP NP NP SEC + LEX ExclusionLimit (Pullulan),, Guard column,, Guard column, Guard column Guard column Guard column,,,,,, (,,) Guard column (,,) Guard column, Guard column Guard column Guard column Line filter Theoretical Plate Number (TP/column),,,,,,,,,,, 9, 9,, 9,,,,, Particle Size (µm) 9 ID x Length (mm) 8. x 8. x. x 8. x 8. x. x 8. x. x. x. x. x. x 8. x 8. x 8. x 8. x 8. x 8. x. x 8. x 8. x. x. x. x. x. x. x. x 8. x. x : SEC (Size exclusion), IEX (Ion exclusion), LEX (Ligand exchage), NP (Normal phase) : Figure in ( ) is estimated value

4 Table. Columns for saccharide analysis () Product Code Product Name Exclusion Limit (Pullulan) Usable Organic Solvents (Max.%) Methanol Acetonitrile DMF Theoretical PlateNumber (TP/column) Particle Size (µm) ID x Length (mm) F9 F9 F9 F9 F9 F9 F9 F9 F98 F9 OHpak SB8 HQ OHpak SB8. HQ OHpak SB8 HQ OHpak SB8 HQ OHpak SB8 HQ OHpak SB8 HQ OHpak SB8M HQ OHpak SBG OHpak SB8 HQ OHpak SB8 G,,,,,,, (,,) (,,) Guard column (,,) Guard column,,,,,,,, 8 8. x 8. x 8. x 8. x 8. x 8. x 8. x. x 8. x. x : Figure in ( ) is estimated value.. Separation Mechanism. Anomer Separation of Saccharides Reducing saccharides can adopt various cyclic structures, as well as a linear structure. In such cases, two tautomers, αtype and βtype, are produced because carbonyl carbon atoms become asymmetric. The relation between αtype and βtype is called anomer. (Fig. ) αd(+)glucose α type βd(+)glucose β type Fig. Anomers Under the conditions in which the conversion rate between the tautomers is low, α and β anomers are separated by the column causing the peak tops to split or widen. (Fig. ) Glucose Galactose Mannose Xylose Arabinose Sample : % each, µl C Anomer separation C Noanomer separation Fig. Influence of anomer separation in saccharide analysis Column : Shodex SUGAR SC Eluent : H O Flow rate :.ml/min Column temp. : (A) C, (B) C Two methods are available to prevent anomer separation during analysis. Analysis at high temperature Analysis under strong alkaline conditions

5 . Shodex SUGAR series A strong feature of the Shodex SUGAR series columns resides in the mechanism of separation in the ligand exchange mode. Ligand exchange refers to a mode of separation based on the interaction (ligand exchange potential) between hydroxyl groups and metal ions to form a complex. Saccharides have a membered (furanose) or membered (pyranose) ring structure, containing a large number of hydroxyl groups. These hydroxyl groups bind together either equatorially or axially with respect to the carbon plane. This conformation of the hydroxyl groups differs depending on the kind of saccharide. Figure shows the relationship between hydroxyl group conformation and counter ion interaction. In Figure (a), three hydroxyl groups form a complex with the metal ion. In Figure (b), only two hydroxyl groups form a complex with the metal ion due to the hydroxyl group conformation. Hence, ligand exchange potential is higher for (a) than for (b). The complex formation potential also differs depending on the kind of metal ion. When using a column of the SUGAR series, analysis should be performed at increased column temperatures to prevent anomer separation. Under strongly alkaline conditions, saccharides are likely to isomerize with the fear of decomposition of polysaccharides. Fig. Difference of counter ion interaction. Shodex Asahipak NHP series Shodex Asahipak NHP series are greatly improved amino columns which not only maintain the high separation performance of the conventional silicabased amino columns, but also solve the problem of declines in retention over time. This is due to stable chemical bonding of polyamine with hydrophilic polymer gel. Other advantages are below. Analysis under moderate conditions (around ph and room temperature) is possible. Sharp, nearsymmetric peaks can be obtained for a wide variety of saccharides. Accurate quantitative determination can be made. A wide range of eluents, such as various buffer solutions, alkaline solutions, or acidic solutions can be used. Alkaline washing of columns is possible. With amino columns, saccharides elute in order of increasing polarity due to the function of normal phase chromatography. Usually, a mixed solvent of acetonitrile and water is used as the eluent. When the mixing ratio of acetonitrile is increased, the polarity of the eluent becomes lower. This results in a stronger interaction between saccharides and the column and a larger elution volume. As NHP columns have weak alkaline amino groups, the condition inside the column is alkaline. This enables saccharides to be analyzed without causing separation of anomers even at room temperature. NHP C Amide column C Sample : mg/ml each,µl. Fructose. Glucose. Sucrose. Maltose 8 C There are columns, called amide columns, which are used for analysis of saccharides under the same elution conditions as those for amino columns. Although amide columns have acrylamide groups introduced, analysis has to be made at high temperature because the acrylamide group is not alkaline. (Fig. ) Fig. Effects of column temperature on elution patterns (comparison with amide column) Column : Shodex Asahipak NHP E (.xmm) Amide column from companya (.xmm) Eluent : CH CN/H O=/ Flow rate :.ml/min Column temp. : C, 8 C

6 NHP columns show good reproducibility for a long time, because the chemical structure of the packing material is stable. With silicabased amino columns, retention of saccharide weakens with the passage of time, resulting in significant widening of every peak. This clearly indicates deterioration of the column. (Fig. ) NHP E Silica based amino column First use After 8hrs First use After hrs Eluent : CH CN/H O=/ Flow rate :.ml/min Column temp. : C Fig. Reproducibility of chromatograms with NHP E column and silicabased amino column. Detection of Saccharides The most commonly used approach for detecting saccharides is the use of the differential refractive index (RI) detector, which is based on refractive index differences between sample components and a mobile phase. Although the RI detector is highly versatile, it exhibits low selectivity and is not suitable for gradient elution. Another drawback is the tendency for lower sensitivity than other types of detectors. In cases where high selectivity and high sensitivity are required, the ultraviolet absorption (UV) detector and the fluorescence (FL) detector are effective. It should be noted, however, that saccharides must be derivatized because they lack a structure for absorbing UV or emitting fluorescence. Other available methods of detection include the mass spectrum (MS) analyzer, the electrochemical (EC) detector which electrochemically detects ionized saccharides under alkaline conditions, and the evaporation light scattering (ELS) detector which detects saccharides by evaporating an eluent and an irradiating laser.. Monosaccharides and Disaccharides The term Saccharide generically refers to simple saccharides, oligosaccharides, polysaccharides, sugar alcohols and the like. Simple saccharides, i.e., monosaccharides and disaccharides, exclude sugar alcohols. Emphasized labeling such as, Very Low Sugar or Low Sugar, and Sugarless is not permitted unless the content of the relevant type of sugar is not more than grams per grams (. g/ ml for refreshing beverages), and less than. grams per grams (. g/ ml for refreshing beverages), respectively. Japan s nutritional facts labeling standards specify the use of an amino column (. mm inner diameter x mm length) to analyze simple saccharides and oligosaccharides (Figure ). Shodex can provide many kinds of columns for saccharide analysis. Carbohydrate Saccharide(or Sugar) Simple saccharide (or Sugar) Monosaccharide, Diretary fiber Oligosaccharide Sugar alcohol Polysaccharide Disaccharide

7 Monosaccharide, Disaccharide Column : A column packed with silica or polymer gel bounded amino (propyl) group (.mmid x mm) Shodex Asahipak NHP E Eluent : CH CN/H O = / Flow rate :. ml/min Detector : RI Column temp. : Room temp. Injection vol. : µl Oligosaccharide Column : A column packed with silica or polymer gel bounded amino (propyl) group (.mmid x mm) Shodex Asahipak NHP E Eluent : CH CN/H O = / Flow rate :. ml/min Detector : RI Column temp. : Room temp. Injection vol. : µl Japan s nutritional facts labeling standards ver. (Japan Health Food Authorization) Fig. Analysis condition of saccharides and oligosaccharides 8 Sample :.% each, µl. Fructose. Sorbose. Galactose. Glucose. Xylobiose. Sucrose. Lactose 8. Maltose Column : Shodex Asahipak NHP E (.mmid x mm) Eluent : CH CN/H O=/ Flow rate :.ml/min Column temp. : C min Fig. Standard saccharides with NHP E Peak height (µv) Sorbitol R =.9999 Glucose R =.9998 Peak height (µv) Sample concentration (mg/ml).. Sample concentration (mg/ml) Table Correlation for other saccharides (R ) Fructose.9998 Sucrose.9999 Lactose.999 Maltose.9998 mesoerythritol.9999 Xylitol.9999 Mannose.9999 Maltitol.999 (.mg/ml~mg/ml) Condeition of Fig. 8 and Table Column : Shodex Asahipak NHP E (.mmid x mm) Eluent : CH CN/H O=/ Flow rate :.ml/min Column temp. : C Fig. 8 Calibration curves with NHP

8 Sample : µl. Fructose. Galactose. Glucose. Sucrose. Lactose (Pretreatment of sample) () Measure g of yogurt into a beaker. () Add ml of pure water to the yogurt. After having stirred the mixture, neutralize it with a w/v% sodium hydroxide aqueous solution. () After minutes of ultrasonic extraction, add pure water until total volume becomes ml. () Pass the solution () with a No. B filter paper. To ml of the filtrate, add the same volume of acetonitrile and stir this mixture. () Pass through a membrane filter (.µm) before injecting into HPLC. min Column : Shodex Asahipak NHP E (.mmid x mm) Eluent : CH CN/H O=/ Flow rate :.ml/min Column temp. : C Fig. 9 Saccharides in sugaradded yogurt Sample : µl. Fructose. Glucose. Sucrose. Maltose (Pretreatment of sample) () Make paste of the sample using a foodprocessor. () Put g of the paste in a beaker and add ml of %(V/V) ethanol. () Extract the substances from the paste using an ultrasonic vibrator for min. () Put the paste and ethanol in a ml messflask and add %(V/V) ethanol to make up a ml solution. () Filtrate the solution with a paper filter(no.b) to remove the paste. () Take a part of filtrated solution and add the same volume of acetonitrile. () Pass through a membrane filter (.µm) before injecting into HPLC. min Column : Shodex Asahipak NHP E (.mmid x mm) Eluent : CHCN/HO=/ Flow rate :.ml/min Column temp. : Room temp.( C) Fig. Saccharides in sweet potato, roasted Sample : µl.% Fructooligosaccharide syrup. Fructose. Glucose. Sucrose. Kestose. Nystose. F FructofranosylDnystose With NHP columns, saccharides elute in order of increasing polarity due to the function of normal phase chromatography. The polarity becomes stronger with longer chains and adsorption becomes stronger. With the analysis of oligosaccharides, high rate of water of solvent is recommended for early elution. min Column : Shodex Asahipak NHP E (.mmid x mm) Eluent : CHCN/HO=/ Flow rate :.ml/min Column temp. : C Fig. Fructooligosaccharide syrup

9 Sample : µl fold diluted Worcestershire sauce. Polysaccharides and Anion. Polysaccharides. Sucrose. Glucose. Fructose With SUGAR KS8, high molecular weights and anionic compounds are eluted first, followed by low molecular weight saccharides. Therefore, KS8 can be used for the analysis of saccharides without any previous desalting of samples such as Worcestershire sauce, which contains large amounts of sodium chloride and organic acids usually. min Column : Shodex SUGAR KS8 (8.mmID x mm) Eluent : HO Flow rate :.ml/min Column temp. : 8 C Fig. Saccharides in worcestershire sauce Sample : Soybean flour. Stachyose. Raffinose. Sucrose. Maltose. Glucose (Sample pretreatment ) () Put the soybean flour in a beaker and add fold pure water. () Extract the substances from the paste into water using an ultrasonic vibrator for min. () Perform ultrafiltration (mw :,). () Pass through a membrane filter (.µm) before injecting into HPLC. min Column : Shodex SUGAR SPG (.mmid x mm) + SP8 (8.mmID x mm) x Eluent : H O Flow rate :.ml/min Column temp. : 8 C Fig. Saccharides in soybean flour Sample : Raspberry jam µl (SC8) Sample : Raspberry jam µl (SC) Sample : Raspberry jam. Oligosaccharide(DP). Oligosaccharide(DP). Oligosaccharide(DP). Oligosaccharide(DP). Maltose. Glucose. Fructose min min Raspberry jam was analyzed. It was found that lots of oligosaccharides are contained in the sample. SUGAR SC or SC8 was used and better separation was obtained by SC8 because of its larger pore size. (Sample pretreatment ) () Put g of raspberry jam in a beaker and add ml of pure water. () Pass through a membrane filter (.µm) before injecting into HPLC. Column : (left); Shodex SUGAR SC (right); Shodex SUGAR SC8 (8.mmID x mm each) Eluent : HO Flow rate :.ml/min Column temp. : 8 C Fig. Saccharides in raspberry jam

10 . Saccharides and Sugar Alcohols Amino columns (.mmid x mm) and ligand exchange mode columns are one of official methods of analysis for sugar alcohols. (Fig. ) Sugar alcohol () Sugar alcohol () Column : A column packed with silica or polymer gel bounded amino (propyl) group (.mmid x mm) Shodex Asahipak NHP E Eluent : CH CN/H O = / Flow rate :.ml/min Detector : RI Column temp. : Room temp. Injection vol. : µl Column : A column packed with sulfonated poly styrene gel (Pb or Caligand) (.88.mmID x mm) Shodex SUGAR SP8, SC Eluent : H O Flow rate :.ml/min Detector : RI Column temp. : 8 C Injection vol. : µl Japan s nutritional facts labeling standards ver. (Japan Health Food Authorization) Fig. Analysis condition of sugar alcohols 8 9 Sample : % each, µl. Sucrose. Glucose. Xylose. Galactose. Fructose. mesoerythritol. Lactitol 8. Mannitol 9. Xylitol. Sorbitol min min Sample : % each, µl. Sucrose. Glucose. Lactitol. Fructose. mesoerythritol. Mannitol. Sorbitol Column Eluent Flow rate Detector Column temp. : Shodex SUGAR SP8 Shodex SUGAR SC (8.mmID x mm each) : H O :.ml/min : Shodex RI : 8 C Fig. Standard saccharides and sugar alcohol with SP8, SC Sample : Confectionery µl. Sorbitol. Xylitol (Sample pretreatment) % aqueous solution of the sample was passed through a filter (.µm), and the same volume of acetonitrile was added. min Column : Shodex Asahipak NHP E (.mmid x mm) Eluent : H O/CH CN=/ Flow rate :.ml/min Column temp. : C Fig. Xylitol in confectionary 8

11 Sample : Apple juice, µl. Sucrose. Glucose. Fructose. Sorbitol (Sample pretreatment) () Take g of apple juice in a beaker, add ml of pure water and neutralize with %(W/V) NaOH aq. cooled with ice. () Apply ultrasonic vibration for min and make ml solution with pure water. () Filtrate with.micron membrane filter. min Column Eluent Flow rate Detector Column temp. : Shodex SUGAR SC (8.mmID x mm) : H O :.ml/min : Shodex RI : 8 C Fig. 8 Apple Juice. Watersoluble Low Molecular Weight Dietary Fibers Dietary fibers are usually quantified by the Prosky method (enzymegravimetric method). However, watersoluble low molecular weight dietary fibers unquantifiable by the Prosky method, the enzyme HPLC method is used. In this analysis, the sample is fractionated into two fractions: dietary fibers (tri and higher saccharides), and mono and disaccharides. Subsequently, the peak area ratio of the dietary fiber fraction and glucose (or internal standard) is determined. Watersoluble low molecular weight dietary fibers Column : Size exclusion chromatography columns (Exclusion limit MW : ca.,) or Ligand exchange columns (Na or Ca type) Eluent : HO Flow rate :.ml/min Detector : RI Column temp. : 8 C Injection vol. : µl Japan s nutritional facts labeling standards ver. (Japanese health and food nutrition society) Fig. 9 Analysis condition of water soluble dietary fiber Using GS HQ, monosaccharides, disaccharides and sugar alcohols elute after the position indicated by the arrow mark and it is easy to divide the two fractions. min Column Eluent Flow rate Detector Column temp. : Shodex Asahipak GS HQ (.mmid x mm) x : H O :.ml/min : Shodex RI : C Fig. Water soluble dietary fiber 9

12 . Saccharides with Amino Acids or Organic Acids Amino acids and organic acids are often contained with saccharides in foods. 8 Sample :.% each, µl. Maltose. Glucose. Fructose. Glutamic acid. Aspartic acid. Threonine. Serine 8. Glutamine 9. Alanine Generally, ligand exchange mode columns are used with aqueous eluent, but the addition of the same cation as the counter ion enables the columns to separate samples under ion exchange mode additionally in addition to the ligand exchange mode. CaSO aqueous solution is used for SUGAR SC as its counter ion is Ca +. This means that ligand exchange occurs between the counter ions of the packing material and the hydroxyl groups of saccharides and ion exchange occurs between the sulfonate groups and the amino groups of the saccharides. 9 min Column : Shodex SUGAR SC (8.mmID x mm) Eluent : mm CaSO (ph.) Flow rate :.ml/min Column temp. : 8 C Fig. Saccharides and amino acids () Sample :. Sucrose. Glucose. Fructose. Taurine. myoinositol Taurine is an amino acid, and it is well known as one of the ingredients added to energy supplement drink recently. 8 min Column : Shodex SUGAR SC (.mmid x mm) Eluent : H O/CH CN=/ Flow rate :.ml/min Column temp. : C Fig. Saccharides and amino acids () Sample :.% each, µl. Maltotriose. Maltose. Glucose. Succinic acid. Lactic acid. Glycerol. Acetic acid 8. EtOH In acidic eluent organic acids are separated with ion exclusion mode and reversed phase mode. Saccharides are separated with SEC. min 8 Column : Shodex SUGAR SH (8.mmID x mm) Eluent : mm H SO aq. Flow rate :.ml/min Column temp. : C Fig. Saccharides and organic acids

13 . Polysaccharides Polysaccharides are used as thickeners/stabilizers to thicken and/or solidify foods and for other purposes. Polysaccharides have a broad range of molecular weights, therefore their analyses are performed not only for quantification but also for molecular weight determination. cp Sample :.% Sodium alginate, µl Mw = x Mn =. x Mw/Mn = 9. Sample :.% Carboxymethylcellulose (CMC), Medium viscosity, µl 8 min cp 8 8 min 8 min Column : Shodex OHpak SB8M HQ (8.mmID x mm) x Eluent :.M NaNO aq. Flow rate :.ml/min Column temp. : C Carboxymethylcellulose was analyzed using OHpak SB8M HQ (a column for GFC separation). Mw and Mn were calculated as pullulan. Column : Shodex OHpak SB8M HQ (8.mmID x mm) x Eluent :.M NaCl aq. Flow rate :.ml/min Column temp. : C Fig. Sodium alginate Fig. Carboxymethylcellulose. Sweeteners Generally food additives are analyzed with reversed phase mode columns: however, saccharides are difficult to analyze because of their high polarity. So columns for saccharide analysis are recommended to analyze saccharides and sweeteners at the same time. Sample : µl..% Acesulfame K..% Sucrose..% Glucose..% Fructose Concentration of acesulfame K is tracee level, it is difficult to detect by RI detection. Saccharides do not have UVabsorption, so there is no peak by UV detection.. min Column : Shodex SUGAR SC (8.mmID x mm) Eluent : mm CaSO aq. Flow rate :.ml/min, UV (nm) Column temp. : 8 C Fig. Acesulfame K and saccharides Table of elution volume of saccharides with each column are shown in appendixa.

14 . Organic Acids Organic acids are weakly acidic substances having carboxyl groups in their molecular structure, and are also referred to as carboxylic acids. When analyzing organic acids, ion exclusion, reversedphase, ion exchange (ion chromatography), size exclusion, and other types of chromatography are used. Shodex columns for organic acid analysis include RSpak KC8 (a combination of ion exclusion mode and reversedphase mode), RSpak DE (polymeric reversedphase column), and IC SI E (anion chromatography column), as well as SUGAR SH (column for analysis of saccharides and organic acids, described in Section ). Table shows a lineup of Shodex packing columns for organic acid analysis. Table. Shodex columns for organic acids analysis Product Code Product Name SeparationMode Theoretical PlateNumber (TP/column) Particle Size (µm) ID x Length (mm) F8 F F F8 F F F F9 F F F F F8 F8 F8 F99 F99 F9 RSpak KC8 RSpak KCG RSpak KCLG RSpak NN8 RSpak NNG RSpak DE RSpak DE RSpak DEL RSpak DES RSpak DEG RSpak DE RSpak DESG SUGAR SH SUGAR SH8 SUGAR SHG IC SI9 E IC SI E IC SI9G IEX+RP IEX+RP IEX+RP IEX+RP IEX+RP RP RP RP RP RP RP RP SEC + IEX SEC + IEX AEC AEC AEC, 9,,,,, 8,,,,, x. x 8. x 8. x. x. x. x. x. x. x. x. x 8. x 8. x. x. x. x. x : SEC (Size exclusion), IEX (Ion exclusion), RP (Reversed phase), AEC (Anion exchange). Separation mechanism Shodex can provide several columns for analysis of organic acids depending on other substances in the sample. (Table ) Table. Column selection for samples Column Separation Mode Sample RSpak KC8 RSpak NN8 RSpak DE SUGAR SH SUGAR SH8 IC SI9 E IC SI E Ion exclusion + Reversed phase Ion exclusion + Reversed phase Reversed phase Size exclusion + Ion exclusion Anion exchange General organic acids Aromatic organic acids General organic acids Organic acids and Sugars Organic acids and Anions

15 . Ion exclusion mode In organic acid analysis, a strong cation exchange resin with sulfogroups bound to the packing surface is used. Weak acids, such as organic acids, exhibit only partial dissociation when dissolved in water. In a dissociated state (negatively charged), the positive charge of the cation is neutralized and excluded by the negative charge of the sulfogroups on the resin surface. There is no concern over organic acid adsorption into the resin. In undissociated state, on the other hand, ion exclusion does not occur; hydrophobic adsorption into the resin substrate occurs instead to allow organic acids to be retained by the resin (Figure ). Undissociated state Dissociated state Ion exclusionh resin Fig. Ion exclusion mode Hence, organic acids are eluted in ascending order of pka (descending order of acidity) and in descending order of polarity. The packing substrate of KC8 is a styrenedivinylbenzene copolymer, therefore it is recommended to use NN8, which has a higher polarity of packing substrate than KC8, when analyzing aromatic organic acids of low polarity. The packing substrate of NN8 is poly(hydroxymethacrylate).. Reversed phase mode The reversedphase mode is the most commonly used separation mode of HPLC. This mode is based on the hydrophobic interaction between the portion of low polarity of the packing and the portion of low polarity of the sample. Hence, elution occurs in descending order of polarity. A key to successful organic acid analysis in reversedphase mode resides in thoroughly suppressing the dissociation of organic acids and reducing the polarity of the sample. Organic acids are generally reported to become nondissociated when the eluent has a ph value lower than the pka by... Anion exchange mode The resin surface is positively charged. The cation on the resin is scrambled by the anion of the component mobile phase and the organic acid anion (anion); elution occurs in ascending order of ionic adsorptivity.. Size exclusion mode Organic acids are eluted in ascending order of molecular size. Larger molecules than pore size of resin are eluted V positon.. Detection of organic acids The functional group shared by organic acids is the carboxyl group, which has a UV absorption band between and nm, thus enabling the use of a UV detector. However, because many other organic substances have the same wavelength band for UV absorption, and also because the molar extinction coefficient is low at to, detection of organic acids is likely to be influenced by impurities. The RI detector lacks selectivity and remains somewhat problematic with regard to sensitivity. Detection using the conductivity detector (CD) is prone to sensitivity variation depending on the kind of organic acid. As a method of selectively detecting organic acids, the postcolumn method using a ph indicator is available which employs a visible absorption spectrum (VIS) detector. (Fig. ) Column : Shodex RSpak KCG (.mmid x mm) + KC8 (8.mmID x mm)x Eluent : mm HClO aq. Flow rate :.ml/min Column temp. : C Sample :. 8ppm Citric acid. 9ppm Tartaric acid. 8ppm Malic acid. ppm Succinic acid. ppm Lactic acid. ppm Formic acid. ppm Acetic acid 8. ppm Levulinic acid 9. ppm Pyroglutamic acid Post column method VIS (nm) CD 89 min 8 9 min RI UV(nm) Fig. Comparison of chromatograms with various detectors 8 min min 8 9 9

16 . Organic acids 9 8 min Sample :. Citric acid. Tartaric acid. Pyrubic acid. Malic acid. Succinic acid. Glyconic acid. Lactic acid 8. Fumaric acid 9. Acetic acid. Levulinic acid. Pyroglutamic acid. Propionic acid. Isobutyric acid. nbutyric acid General organic acids were analyzed with postcolumn method. After separation with KC8 column, reaction reagent was added and detected with nm. Column : Shodex RSpak KC8 (8.mmID x mm) x Eluent : mm HClO aq. Reagent : fold diluted STR (for postcolumn method) Flow rate :.ml/min Detector : VIS (nm) Column temp. : C Fig.. Analysis of organic acids with postcolumn method Eluent : mm HCIO 8 9 Eluent : mm HCIO /CH CN=9/ min Sample :. Succinic acid. Lactic acid. Formic acid. Acetic acid. Propionic acid. Isobutyric acid. nbutyric acid 8. Isovaleric acid 9. nvaleric acid The elution time of organic acids with strong hydrophobicity can be shortened by adding % acetonitrile to the eluent. Column : Shodex RSpak KCLG (8.mmID x mm) + KC8 (8.mmID x mm) x Reagent : fold diluted STR (for postcolumn method) Flow rate : (Eluent);.mL/min, (Reagant);.mL/min Detector : VIS (nm) Column temp. : C Fig. Effect of elution of organic acids with addition of acetonitrile Post column method VIS (nm) µl of Soy sauce UV(nm) µl of Soy sauce 8 min min CD RI µl of Soy sauce µl of Soy sauce 8 Sample : Soy sauce. Citric acid. Pyruvic acid. Malic acid. Succinic acid. Lactic acid. Formic acid. Acetic acid 8. Pyroglutamic acid For actual samples such as foods containing amino acids, sugars, NaCl, ions, organic substances, etc., the postcolumn method is recommended because it enables highly selective analysis. UV detection ( nm) detects many organic substances other than organic acids, and conductivity detection produces a major peak of Cl ions excluded at a position corresponding to nearly minutes; therefore, the quantifiability of organic acids is affected with these methods. In the case of RI detection, organic acids are difficult to quantify because almost all components, including sugars, are detected. 8 min 8 min Column : Shodex RSpak KCG (.mmid x mm) + KC8 (8.mmID x mm) x Eluent : mm HClO aq. Flow rate :.ml/min Column temp. : C Fig. Comparison of results of soy sauce with various detectors

17 High quallity wine 8 Table wine Sample : Wine, µl. Citric acid. Tartaric acid. Malic acid. Succinic acid. Lactic acid. Fumaric acid. Acetic acid 8. Pyroglutamic acid min min Grapes are rich in malic acid. Wine reportedly becomes mellow as a result of malic acid conversion to lactic acid by the action of enzymes during malolactic fermentation. Vintage items prove to have almost all malic acid converted to lactic acid. Column : Shodex RSpak KCLG (8.mmID x mm) + KC8 (8.mmID x mm) x Eluent : mm HClO aq. Reagent : / STR (for postcolumn method) Flow rate : (Eluent);.mL/min (Reagent);.mL/min Detector : VIS(nm) Column temp. : C Fig. Organic acids in white wine Sample :. Glyoxylic acid,.8mg/ml. Tartaric acid,.9mg/ml. Malic acid,.mg/ml. Lactic acid, µl/ml. Malonic acid,.9mg/ml. Acetic acid, µl/ml. Succinic acid,.mg/ml 8. Levulinic acid,.9mg/ml 9. Propionic acid, µl/ml When water or % buffer is used as the eluent, the column life of ODS columns is usually shortened. Since the packing material of RSpak DE is chemically stable, long column life and stable analysis results can be obtained even when water or % buffer was used as the eluent min Column : Shodex RSpak DE (.mmid x mm) Eluent : mm H PO aq. Flow rate :.ml/min Column temp. : C Fig. Organic acids (reversed phase mode) Sample : fold diluted Beer, µl. Acetic acid. H PO, Succinic acid, Pyroglutamic acid. Lactic acid, Pyruvic acid. Cl. Malic acid, Br. NO. Oxalic acid 8. Citric acid 9. SO. Tartaric acid The eluent used here is suitable for the simultaneous analysis of inorganic anions and dibasic and tribasic organic acids using IC IA. Organic acids are contained in beer and they interfere with the quantification of anions. Cl, NO and SO can be separated without such interference of organic acids. Organic acids, oxalic acid and citric acid also can be analyzed without interference. 9 8 min Column : Shodex IC IA (.mmid x mm) Eluent :.mm Phthalic acid +.8mM Tris(hydroxymethyl) aminomethane(ph.) + mm Boric acid Flow rate :.ml/min Detector : Shodex CD Column temp. : C Fig. 8 Organic acids and anions in beer (anion exchange mode) Table of elution volume of organic acids with each column are shown in appendixb, C and D.

18 . Vitamins There are two type of vitamins, fatsoluble vitamin and watersoluble vitamin. (Fig. ) It is possible to analyze vitamins simultaneously, though several official methods show the analysis methods respectively. The major analysis by HPLC are reversed phase or normal phase, additionally size exclusion chromatography can be applied. Table shows the lineup of Shodex packed columns for vitamin analysis with separation modes.. Fatsoluble Vitamins A D E K Retinol, βcarotene Ergocalciferol (D ), Cholecalciferol (D ) α, β, γ, δtocopherol Phylloquinone (K ), Menaquinone (K ), Menadione (K ) Watersoluble Vitamins B B B B B B B B T C H M Thiamin Riboflavin Niacin (Nicotinic acid), Niacinamide (Niacinamide) Pantothenic acid Pyridoxine, Pyridoxal, Pyridoxiamin Cyanocobalamin Orotic acid Carnitine Ascorbic acid Biotin Folic acid Fig. Categories of vitamins Table Shodex columns for vitamin analysis (Absorption and Partition) Product Code Product Name Theoretical Plate Number (TP/column) Particle Size (µm) ID x Length (mm) F F F F F F F F F F9 F F F F9 F F F F F F F F F F F Asahipak ODP D Asahipak ODP E Asahipak ODP D Asahipak ODP E Asahipak ODPG A Asahipak ODP B Asahipak ODP D Asahipak ODP E Asahipak ODPG A Asahipak ODP D Asahipak ODPG A RSpak DE RSpak DE RSpak DEL RSpak DES RSpak DEG RSpak DE RSpak DESG Asahipak NHP D Asahipak NHP E Asahipak NHPG A Asahipak NHP D Asahipak NHPG A Silica SIL D Silica SIL E,, 9,,, 9,,,,,,, 8,,,, 9,,. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x

19 ( Size Exclusion ) Product code Product Name Theoretical plate number (TP/column) Particle Size (µm) Exclusion Limit (Polystyrene) ID x Length (mm) F8 GPC KF8 8,, 8. x F GPC KFG 8. x ( Multimode ) Product code Product Name Theoretical plate number (TP/column) Particle Size (µm) ID x Length (mm) F8 RSpak NN8 9, 8. x F8 RSpak NN,. x F RSpak NNG. x F8 RSpak NN,. x. FatSoluble Vitamins 8 Sample : µl. Menadione (Vitamin K ). Retinol (Vitamin A). Retinol acetate (Vitamin A acetate). Menadione (Vitamin D ). Cholecalciferol (Vitamin D ). αtocopherol acetate (Vitamin E acetate). αtocopherol (Vitamin E) 8. Phylloquinone (Vitamin K ) min Column : Shodex Asahipak ODP E (.mmid x mm) Eluent : CH CN/CH OH=/ Flow rate :.ml/min Detector : UV(8nm) Column temp. : C Fig. Simultaneous analysis of fatsoluble vitamins (reversed phase mode) Sample : µl. Retinol palmitate (Vitamin A palmitate). αtocopherol (Vitamin E). Cholecalciferol (Vitamin D ). Menadione (Vitamin K ) min Column : Shodex GPC KF8 (8.mmID x mm) x Eluent : THF Flow rate :.ml/min Detector : UV(8nm) Column temp. : C Fig. Simultaneous analysis of fatsoluble vitamins (size exclusion mode)

20 Sample :.% Margarine, µl. Retinol palmitate (Vitamin A palmitate) The Vitamin A palmitate was analyzed using GPC KF8. Wave length of nm at which it has maximum UV absorption was used for the detection. Only dissolving the sample in the eluent(thf) and filtrating it using a disposable filter(.µm), vitamin A palmitate can be analyzed without any other pretreatment like saponification. min Column : Shodex GPC KF8 (8.mmID x mm) x Eluent : THF Flow rate :.ml/min Detector : UV (nm) Column temp. : C Fig. Vitamin A palmitate in margarine (size exclusion mode) Sample :.µg/ml each, µl. Ergocalciferol (Vitamin D ). Cholecaciferol (Vitamin D ) Vitamin D and D were separated using Asahipak ODP D ( polymerbased column ) by reversed phase mode. Since the usable ph range of the ODP column is very wide and it can be washed with both acidic and alkaline solvents, it is very durable even if it is used for crude samples. Column : Shodex Asahipak ODP D (.mmid x mm) Eluent : CH CN/CH OH=9/ Flow rate :.ml/min min Detector : UV (nm) Column temp. : C Fig. Vitamin D and D (reversed phase mode) Sample : Tocopherol. αtocopherol. βtocopherol. γtocopherol. δtocopherol Alpha, beta, gamma and deltatocopherol were separated by normal phase mode using the column with a mixture of nhexane/isopropanol as the eluent. min Column : Shodex Silica SIL D (.mmid x mm) Eluent : nhexane/propanol=99./. Flow rate :.ml/min Detector : UV (8nm) Column temp. : C Fig. Vitamin E (normal phase mode) 8

21 . WaterSoluble Vitamins min Sample : µl. Ascorbic acid (Vitamin C). Niacin (Vitamin B ). Pyridoxine (Vitamin B ). Niacinamide (Vitamin B ). Thiamine (Vitamin B ). Cyanocobalamin (Vitamin B ). Riboflavin (Vitamin B ) Column : Shodex RSpak DE (.mmid x mm) Eluent : (A); /M Potasium phosphate buffer(ph.8) /CH CN=98/ (B); H O/CH CN=/ min to min, % (A) min to min, % (A) to % (B) After min, % (B) Flow rate :.ml/min Detector : Shodex UV (nm) Column temp. : C Fig. Simultaneous analysis of watersoluble vitamins (reversed phase mode) Sample :.% each, µl. Pantothenic acid. Orotic acid. Pyridoxine HCl (Vitamin B ). Nicotinamide. Vitamin B. Vitamin B The NN8 is a multimode column (reversed phase + cation exchange). Using NN8, isocratic analysis can be carried out, and it does not require an ion pair reagent. min Column : Shodex RSpak NN8 (8.mmID x mm) Eluent : mm KH PO + mm Na HPO + % CH OH Flow rate :.ml/min Detector : UV (nm) Column temp. : Room temp. Fig. 8 Simultaneous analysis of watersoluble vitamins (multi mode) Sample : ng/µl Thiamine hydrochloride, µl Fluorescence intensity The total quantity of vitamin B was measured using RSpak NN with a phosphate buffer eluent. The vitamin B separated by the column is converted into thiochrome using a postcolumn reagent and detected by a fluorescence detector. This method was recommended by a Japanese standard, "Standard Methods of Analysis for Hygienic Chemists with Commentary" (99). min. Vitamin B (thiamine) Vitamin B (ng) Thlochrome Reagent Column : Shodex RSpak NN (.mmid x mm) Eluent : mm KH PO + mm Na HPO aq. Reagent :.% K Fe(CN) + % NaOH Flow rate :.ml/min Detector : Fluorescence (Ex. nm, Em. nm) Column temp. : C Fig. 9 Vitamin B (multi mode) 9

22 Members of the vitamin B family are difficult to separate due to structural similarity. Although the usual method of separation employs an ion pair reagent, analysis in reversedphase mode is possible on the basis of slight differences in hydrophobicity among individual samples, provided that the acidity is high, for example, ph.. Actually, three ingredients were separated in about minutes using the Asahipak ODP polymeric reversedphase column, which is stable even at a ph of.. min Fig. Vitamin B (reversed phase mode) Column : Shodex Asahipak ODP D (.mmid x mm) Eluent : mm Sodium phosphate buffer(ph.) Flow rate :.ml/min Detector : UV (nm) Column temp. : C O=C HOC O HOC HC HCOH CH OH Erythorbic acid O=C HOC O HOC HC OHCH Sample : µg/n. Ery. Aso CH OH LAsorbic acid Amino columns or silica columns are usually used for the analysis of ascorbic acid. Using amino columns is particularly wide spread because isoascorbic acid (erythorbic acid) as well as ascorbic acid can be analyzed simultaneously. However, conventional silicabased amino columns are not chemically stable, so their life time is not long. Asahipak NHP E has long life time, since it is a polymerbased column and stable. Column : Shodex Asahipak NHP E (.mmid x mm) Eluent : mm NaH PO +mm H PO aq. /CH CN=/8 Flow rate :.ml/min Detector : UV(nm) Column temp. : C min Equilibration : After passing mm phosphoric acid in the column by.ml/min for h, it replaces by eluent. Fig. Vitamin C (nomal phase mode) Sample : Carnitine (Vitamin B T ) Carnitine (Vitamin B T ) standard was separated using RSpak NN 8 (multimode). Main peak was observed at min and impurity peaks were observed at 8 to min. min Column : Shodex RSpak NN8 (8.mmID x mm) Eluent :.M KH PO aq. Flow rate :.ml/min Column temp. : Room temp. Fig. Vitamin B T (multi mode)

23 . Other ingredients in food. Higher Fatty Acids Sample : C n H n+cooh, µl. Capric acid (n=9). Lauric acid (n=). Myristic acid (n=). Palmitic acid (n=). Stearic acid (n=). Arachidic acid, Eicosanoic acid (n=9). Behenic acid, Docosanoic acid (n=) Column Eluent : Shodex RSpak DE (.mmid x mm) : CH CN/THF/H O=// Flow rate :.ml/min 8 8 min Column temp. : C Fig. Higher fatty acids () Sample :. EPA (Eicosapentaenoic acid). DHA (Docosahexaenoic acid). Arachidonic acid. Linoleic acid EPA and DHA, essential fatty acids rich in blue fish and the like, are reported to be effective in preventing thrombosis and enhancing brain function, respectively. min Column : Shodex ODSpak F (.mmid x mm) Eluent : H O/CH CN=/8 Flow rate :.ml/min Column temp. : C Fig. Higher fatty acids () Table Shodex columns for higher fatty acid analysis (Absorption and Partition) Product Code Product Name Theoretical Plate Number (TP/column) Particle Size (µm) Carbon Content (%) ID x Length (mm) F RSpak DE,. x F RSpak DE,. x F9 RSpak DEL,. x F RSpak DES,. x F RSpak DEG. x F RSpak DE 8,. x F RSpak DESG. x F ODSpak F 8,. x F ODSpak F,. x F ODSpak F/S,. x

24 . Nucleic Acids as Taste Components 8 9 Sample : min IMP, GMP, and AMP (nucleotides), representative umami ingredients (tasty nucleic acids), and all of their metabolites were analyzed by isocratic elution using the Asahipak GS E column. Not only the size exclusion mode (main mode), but also the reversedphase and ion exchange modes are available therefore, the GS E column is capable of completely separating within ca. min a total of nine ingredients (corresponding nucleosides and some bases) from IMP, GMP, AMP and other nucleotides. Column : Shodex Asahipak GS E (.mmid x mm) Eluent : mm Sodium phosphate buffer (ph.) Flow rate :.ml/min Detector : UV (nm) Column temp. : C Fig. Nucleic acids as taste components Table Shodex columns for tasting compounds analysis (Multimode) Product Code Product Name ID x Length (mm) F F9 Asahipak GS E Asahipak GSG B. x. x

25 . Amino acids Sample :.nmol each, µl Column : Shodex CXpak PS (.mmid x mm) Eluent : No.;.M Sodium citrate buffer(ph.) No.;.M Sodium citrate buffer(ph.) No.;.M Sodium citrate buffer(ph.) No.;.M Sodium citrate buffer(ph.9) No.;.M NaOH + % C H OH (Rinse solution) Low pressure gradient: min, No..min, No..min, No..min, No..min, No. Flow rate :.ml/min Detector : VIS (nm) (Ninhydrin reaction;.ml/min, C) Column temp. : C Fig. Amino acids (strong cation exchange mode) Sample : µl. Aspartic acid. Glycine. Alanine. Valine. Methionine. Isoleucine Acidic amino acids, such as aspartic acid, are separated by ion exclusion mode and elute faster. Neutral substances are separated by reversed phase and alkaline amino acids are separated by a mixture of reversed phase (in case of hydrophobic structure) and ion exchange mode. Therefore, amino acids basically elute in the following order: first the acidic amino acids, second the neutral amino acids and finally the alkaline amino acids. 8 min Column : Shodex RSpak NN8 (8.mmID x mm) Eluent : mm H PO aq. Flow rate :.ml/min Column temp. : C Fig. Amino acids (multimode) Table Shodex columns for amino acids analysis (Strong cation exchange) Product Code Product Name SeparationMode Particle Size (µm) ID x Length (mm) F F CXpak PS CXpak PG Strong cationexchange. x. x Table Shodex columns for amino acids analysis (Multimode) Product Code Product Name SeparationMode Particle Size (µm) ID x Length (mm) F8 F8 F F8 RSpak NN8 RSpak NN RSpak NNG RSpak NN Ion exclusion + Ion exchange + Reversed phase 8. x. x. x. x

26 Appendix A. List of elution volume of saccharides Elution Volume (ml) NAcetylαDglucosamine D(+)Arabinose DArabitol Aspartame DeoxyDglucose Difructose anhydride III Dulcitol mesoerythritol Ethanol FructofuranosylDnystose D()Fructose D(+)Fucose D(+)Galactose 'Galactosyllactose αdgalacturonic acid Gentiobiose Glucose Glycerol Glycyrrhizin ic myoinositol Isomaltose Isomaltotriose Kestose Kojibiose Lactitol Lactose Lactosylfructoside Lactulose Maltitol Maltoheptaose Maltohexaose Maltopentaose Maltose Maltotriose Mannitol DMannose D(+)Melezitose Melibiose MethylαDmannopyranoside Nystose Substances SP8 Pb + Palatinit peaks peaks ( ) cannot be detected ( ) cannot be separated from solvent peak SC Ca SUGAR series Asahipak Asahipak KS8 Na SZ Zn peaks SC Ca + NHP E peaks GS HQ x

27 Elution Volume (ml) Substances SP8 Pb + SC Ca + SUGAR series Asahipak Asahipak KS8 Na + SZ Zn + SC Ca + NHP E GS HQ x Palatinose mL. Panose LPhenylalanine. D(+)Raffinose D(+)Rhamnose D()Ribose Rutinose Saccharin sodium D()Sorbitol D(+)Sorbose Stachyose Stevioside.. Sucrose αdtalose Theanderose peaks peaks peaks peaks peaks Trehalose Trehalulose Xylitol Xylobiose D(+)Xylose DXylulose ( ) cannot be detected ( ) cannot be separated from solvent peak Column : Shodex SUGAR SP8, SC, KS8 (8.mmID x mm each) Eluent : H O Flow rate :.ml/min Column temp. : 8 C Column : Shodex SUGAR SZ (.mmid x mm) Eluent : H O/CH CN=/ Flow rate :.ml/min Column temp. : C Column : Shodex Asahipak NHP E (.mmid x mm) Eluent : H O/CH CN=/ Flow rate :.ml/min Column temp. : C Column : Shodex Asahipak GS HQ (.mmid x mm) x Eluent : H O Flow rate :.ml/min Column temp. : C Column : Shodex SUGAR SC (.mmid x mm) Eluent : H O/CH CN=/ Flow rate :.ml/min Column temp. : C

28 B. List of elution volume of saccharides and organic acids ( SH) Sample Eluent Sample Eluent mm mm mm mm Oxalic acid.. myoinositol.. Stachyose.. Malic acid.. Isomaltotriose.. DMannose.. Panose.. D(+)Galactose.. Maltotriose.. D(+)Xylose.. Gentiobiose.. Malonic acid.. Isomaltose.8.8 D()Fructose.. Melibiose.9.9 transaconitic acid.. Kojibiose.9.9 Mannitol.. Maltose.9.9 D()Sorbitol.. Trehalose.9.9 D(+)Rhamnose.. Nigerose.9.9 D(+)Arabinose.. Palatinit.. D()Ribose.8.8 Palatinose..9 DArabitol.8.9 Trehalulose.. Xylitol Lactose.. D(+)Fucose Xylobiose.. Succinic acid Maltitol.. mesoerythritol DGlucuronic acid.. NAcetylαDglucosamine Citric acid.. DLLactic acid αketoglutaric acid..9 NAcetylαDgalactosamine Maleic acid.. Formic acid Lactitol.. Acetic acid.. NAcetylneuramic acid.. Adipic acid.. Tartaric acid.. Mesaconic acid.. Glucose.8.8 LPyroglutamic acid.. Pyruvic acid.9. Propionic acid.. Sorbose.9.9 Ethanol.. Column : Shodex SUGAR SH (8.mmID x mm) Eluent : mm or mm H SO aq. Flow rate :.ml/min Column temp. : C

29 C. List of elution volume of organic acids (KC8) Elution Volume (ml) Substance mm HCIO mm HCIO mm HCIO mm HCIO Oxalic acid Maleic acid alphaketoglutaric acid...9. Oxalacetic acid...8. Pyruvic acid...8. Ketogluconic acid.... Phosphoric acid.... Citric acid Tartaric acid.... Isocitric acid Glucuronic acid....8 transaconitic acid.... Malonic acid.... Galacturonic acid.8... Gluconic acid...8. Glyoxylic acid Malic acid.... alphahydroxygltaric acid..9.. Ascorbic acid Fumaric acid....8 Succinic acid...8. Glycolic acid..9.. Itaconic acid Lactic acid.... Hydroxyisobutyric acid..8.. Adipic acid betahydroxypropionic acid.9... Formic acid Mesaconic acid Uric acid Pyroglutamic acid Acetic acid Levulinic acid.... Propionic acid.... CO,HCO.... Isobutyric acid.... nbutyric acid.... Isovaleric acid.9... nvaleric acid Column : Shodex RSpak KC8 (8.mmID x mm) x Eluent : HClO aq. Flow rate :.ml/min Column temp. : C

30 D. List of elution volume of organic acids (KC8) Elution Volume (ml) Substance C C C C C 8 C Oxalic acid Maleic acid Phosphoric acid αketoglutaric acid Ketogluconic acid Glucuronic acid..... Pyruvic acid Citric acid Oxalacetic acid Isocitric acid Tartaric acid Galacturonic acid Gluconic acid Malonic acid transaconitic acid Glyoxylic acid Malic acid Ascorbic acid αhydroxygltaric acid Succinic acid Glycolic acid Lactic acid Hydroxyisobutyric acid βhydroxypropionic acid Itaconic acid Formic acid Fumaric acid Acetic acid Uric acid Levulinic acid Pyroglutamic acid Adipic acid Propionic acid Mesaconic acid CO,HCO Isobutyric acid nbutyric acid Isovaleric acid nvaleric acid Column : Shodex RSpak KC8 (8.mmID x mm) x Eluent : mm HClO aq. Flow rate :.ml/min 8

31 Notice. Please read the instruction manual accompanying the product in its entirety before using the NHP column.. The specifications for the products are subject to change without notice for purposes of improvement.. No guarantee is offered to figures in this technical paper; those figures should be used just as reference.. Even if no precautions are given in the instruction manual as to the safety or danger of reagents and chemical products, make sure that in handling the products, the usual precautions are taken.. The products described herein are not designed for use in clinical examinations in the medical area.

32 Welcome to Shodex provides information of new products and new analysis technologies by . If you are interested in receiving newsletters by , please join our "Chromato Mail" newsletter list, through our website. If you have any question regarding this technical notebook, we welcome your questions through the website below. Manufactured by Shodex (Separation & HPLC) Group, Ogimachi, Kawasakiku, Kawasaki, Kanagawa 8 JAPAN TEL: +89 FAX: +899 Shodex Sales offices (NORTH AMERICA) Shodex / Showa Denko America, Inc. (EUROPE) Shodex / Showa Denko Europe GmbH (ASIA & PACIFIC) Shodex / Shoko Co., LTD. 89 Fifth Avenue, 8th Floor, New York, NY USA Tel : + Fax : + [email protected] KonradZusePlatz D889 Munich, Germany Tel : +9()89999 Fax : +9() [email protected], Shibakohen chome, Minatoku, Tokyo, 8, Japan Tel : +8()9 Fax : +8()98 [email protected] TNE.No.E.()..DEC.TR