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USER`S MANUAL

1. For analysis the use of either 4N Methane Sulfonic Acid Dodecanethiol HCl or Thiogly colic acid has been shown to generate some useful results for TRP however yields tend to be low for all of these methods For CYS analysis reduction and alkylation to generate either carboxymethyl cysteine or pyridylethyl cysteine are the preferred methods for detection for EZ faast procedure cysteic acid cannot be detected by EZ faast Procedures for useful hydrolysis methods can by found in the following references Stein and Moore Methods in Enzymology 6 pp 819 831 1963 Tarr in Microcharacterization of Proteins Shively ed Humana Press 1986 e Miedel et al J Biochem Biophys Methods 18 pp 37 52 1989 Strydom et al in Techniques in Protein Chemistry IV Angeletti ed 1993 Jones et al J Liquid Chromatography 4 pp 454 486 1981 For additional information contact your Phenomenex Technical Representative 3 4 Sample Preparation by SPE and Derivatization Please first refer to section 3 2 if you have not prepared fresh Elution Medium yet The fresh ly prepared Eluting Medium vial may be placed in one of the empty slots in the reagent tray 1 For each sample line up one glass sample preparation vial in the vial rack Figure 2 Be aware of some variability in vial opening and sorbent tip dimensions which may prevent the tip from reaching to the bottom of the sample preparation vial Note Droplets of liquid in
2. gt breaking with tradition in Ca Rc 1x EZ aast Amino Acid Analysis of Protein Hydrolysates by GC FID or GC NPD For Part Number REF KG0 7167 T 17 2 2 5 2 a 2 S lt 3 lt lt T C mma 9 e 5 phenomenex breaking with tradition Phenomenex Ltd Deutschland Zeppelinstr 5 411 Madrid Ave 63741 Aschaffenburg ud Torrance CA 90501 1430 Deutschland USA EZ faast User s Manual The following is a description of the symbols used in the EZ faast manuals on EZ faast packaging and EZ faast kit components Symbol for In Vitro Diagnostic Medical Device Symbol for Manufacturer Symbol for Authorised Representative In The European Community ud T Symbol for Use By and or Expiration Date 9 Symbol for Batch Code and or Lot Number Symbol for Catalogue Number Symbol for Serial Number Symbol for Flammable Substances Symbol for Irritating or Harmful Substances oh Me Symbol for Corrosive Substances TABLE or CONTENTS ee 1 VervigWi s reete eerie ree 2 Sample Preparation 5 Gas Chromatograph
3. pipette Phenomenex P N AH0 5967 or equivalent 10 100yL pipette SoftGrip pipette Phenomenex P N 5966 or equivalent e Pipette tips Phenex Phenomenex P N 0 5917 200pL and AH0 5920 1mL or equivalent Vortex Vials of an appropriate volume with caps see section 3 2 Pasteur pipettes for sample transfer see section 3 4 step 15 Container for proper waste disposal Reagent and supplies for Protein Hydrolysis Septa Auto Sep T 11mm SGE P N 041883 fits Agilent or Carlo Erba instruments or equivalent User s Manual 2 0 Overview 2 1 Overview The EZ faast amino acid analysis procedure consists of a solid phase extraction step fol lowed by derivatization and liquid liquid extraction derivatized samples are quickly analyzed by gas chromatography with FID or NPD detection The solid phase extraction is performed via a sorbent packed tip that binds amino acids while allowing interfering compounds to flow through Amino acids on sorbent are then extruded into the sample vial and quickly derivatized with reagent at room temperature in aqueous solution Derivatized amino acids concomitantly migrate to the organic layer for additional separation from interfering compounds An aliquot from the organic is analyzed on a GC system with either a FID or NPD detector Total sample preparation time takes around 8 minutes and analysis is performed in around 7 minutes for a total start to f
4. C C or polar amino acids SER HYP Improper liner Use deactivated liners see section 4 4 Analyze samples only after making priming injections Decrease in peak height for early eluting amino acids Sample too concentrated the capacity of the SPE tip exceeded Use less sample for preparation see section 3 4 step 2 Constantly moni tor the area for the IS peak Volatile derivatives evaporated during removal of residual reagents with nitrogen gas Adjust nitrogen flow to minimal stop evaporation before sample gets completely dry Low peak height for late eluting derivatives Carrier gas leak Check instrument for leaks reinstall the column and check o ring on liner Peak height for IS Norvaline lower compared to other early eluting amino acids in the standard mix Pipetting error Recalibrate pipette used for dispensing Reagent 1 Constantly monitor area for IS Ghost peaks Pipette tips used for dispensing reagents or for transferring prepared samples may be a source of con taminant peaks Use polypropylene pipette tips of appropriate quality see ordering info on page 15 Always use the Microdispenser for dispensing Reagents 4 5 and 6 Use Pasteur pipettes to transfer the reconstituted sample into a glass insert Interfering peaks may result from extracted contaminants in plastic sample preparation vials Use the vials provided in the kit For autosampler vials order
5. LC MS Protein Hydrolysate Kit with 250 x 2 0mm column LC MS Protein Hydrolysate Kit with 250 x 3 0mm column GC Free Physiological Amino Acid Standards SD1 SD2 amp 03 2mL vial x 2 GC Protein Hydrolysate Standard SD 2mL vial x 2 LC MS Free Physiological Amino Acid Standards for LC SD1 SD2 amp SD3 2mL vial x 2 LC MS Protein Hydrolysate Standard SD 2mL vial x 2 Order No KG0 7165 KG0 7166 KG0 7167 KG0 7168 KH0 7337 KH0 7338 KH0 7339 KH0 7340 0 7184 0 7263 AL0 7500 AL0 7501 Unit ea ea ea ea ea ea ea ea ea ea ea ea www phenomenex com amp 1 4 e 7 Sa E products are available worldwide For the distributor in your coi milk 7 phenomenex S contact Phenomenex USA International Department by telephone fax or e iternationalOphenomenex com breaking with tradition p USA Puerto Rico Canada France United Kingdom Germany New Zealand Australia mail 411 Madrid Ave 273 Sierra Morena 411 Madrid Ave Parc des Grillons Bat3 Queens Avenue Queens Avenue Zeppelinstr 5 P 0 Box 31601 PO Box 4084 Torrance CA Suite 104 Torrance CA 60 route de Sartrouville Hurdsfield Ind Est Hurdsfield Ind Est 63741 Aschaffenburg Milford Lane Cove NSW 2066 90501 1430 San Juan 90501 1430 78232 Le Pecq Cedex Macclesfield Cheshire Macclesfield Cheshire Germany Auckland Australia USA Puerto Rico 00926 USA France SK10 2BN UK SK10 2BN UK New
6. Phenomenex P N AH0 4610 and for inserts 0 4604 Early deterioration of column performance Residual sample preparation reagents degrade column stationary phase Make sure to sample only the upper organic layer Remove the first 20cm of column and re install Interfering peaks drug metabolites Physiological sample anticoagulants like citrate or citric acid may inter fere in the amino acid profile Samples collected with EDTA and heparin anticoagulants are recom mended Confirm peak identity based on mass spectrum User s Manual 6 0 STORAGE AND STABILITY Some amino acids are chemically unstable in physiological fluids e g progressive decline of plasma cystine in time and also in standard mixtures Keep samples and standard mixtures in the freezer Old amino acid standard mixtures and mixtures which have not been stored properly should not be used for instrument calibration Order fresh mixture from Phenomenex see ordering info in Phenomenex catalog Samples prepared for gas chromatographic analysis following the procedure outlined in this manual may be stored for several days in a freezer before analysis For longer storage we recommend that samples be desiccated with anhydrous sodium sulfate vials be capped and placed in the freezer Since sample preparation is expeditious with this procedure we recom mend analyzing samples prepared freshly Samples prepared during
7. Zealand tel 310 212 0555 800 541 HPLC 800 543 3681 01 30 09 21 10 01625 501367 01 247 5405 06021 58830 0 09 4780951 02 9428 6444 fax 310 328 7768 310 328 7768 310 328 7768 01 30 09 21 11 01625 501796 44 1625 501796 06021 58830 11 09 4780952 02 9428 6445 email info info info franceinfoG ukinfo eireinfo anfrage info info phenomenex com phenomenex com phenomenex com phenomenex com phenomenex com phenomenex com phenomenex co nz phenomenex com au
8. the day may be left on the autosampler tray at room temperature to be analyzed during the night or the next day 7 0 CLEANING AND or SUPPLIES The Drummond Dialamatic Microdispenser should be flushed with isopropanol acetone approx 1 1 at the end of the day Please review the Drummond Microdispenser users manual for further care and use notes The same organic mix is recommended as wash for both manual syringes and autosamplers Plastic syringes used for sample clean up by SPE can be cleaned by flushing with propanol water 1 2 v v mixture Always tightly cap the reagent bottles when not in use in order to avoid solvent evapora tion and alteration of reagent composition Cover the racks holding sorbent tips when not in use to prevent contamination 8 0 Quatity AsSURANCE All components of the EZ faast GC Amino Acid Analysis Kit are subjected to rigorous quality control testing These measures help to ensure the best results If poor results occur please contact your Phenomenex technical consultant or distributor 9 0 Propuct Limitations Phenomenex Analyte Specific Reagent products are not intended for clinical use Because they are not intended for clinical use no claim or representation is made or intended for their Clinical use including but not limited to diagnostic prognostic therapeutic or blood banking It is the user s responsibility to validate the performance of Phenomenex products for any par ticular use sin
9. 10 Tyrosine TYR Y 206 107 164 107 0 4 0 2 Diaminopimelic DAPA 256 168 Proline hydroxyproline dipeptide 156 186 156 114 0 9 10 Tryptophan RP 130 130 0 4 0 1 Lysine alanine dipeptide LYS ALA 170 224 153 Dopamine DA 179 136 123 3 Nitrotyrosine 152 209 Aspartame 302 Cystathionine CTH 203 272 146 114 4 10 3 4 Dihydroxyphenylalanine DOPA 222 123 Cystine c c Cys 2 248 216 114 173 4 10 Serotonin SRO 146 288 348 Homocystine HC CH Hcys 2 230 188 128 Arginino succinic acid ARG SUC 441 326 Ethionine ETH 203 291 143 LODs were determined for amino acids included in standard mixtures provided with the kit Several amino acids coelute under the chromatographic conditions specified in the user manual e g GABA amp SER 2 3 Storage and Stability Store Reagents 1 3B and 4 at 4 C do not freeze Store amino acid standard solutions in the freezer All other components may be stored at room temperature For your convenience the bottom of the reagent box has been designed as a tray which can be easily lifted from the work station and placed in the refrigerator when the kit is not in use for an extended period of time All components are guaranteed for 12 months or more see label on bottle vial from the date of purchase when stored at recommended temperatures and used as described in this manual Please review the Instruction Manual included with the Drummond Dialamatic Microdispenser for recommended us
10. 47 174 147 04 0 1 Aspartic acid ASP D 216 130 216 130 0 9 0 1 Methionine MET M 203 277 101 203 129 0 9 0 2 3 Hydroxyproline 3HYP 172 259 130 4 Hydroxyproline 4HYP OHPro 172 86 172 86 2 0 2 Phenyl glycine PHE GLY Seleno methionine Se MET Glutamic acid GLU E 230 170 84 142 2 0 2 Phenylalanine PHE F 206 190 147 128 91 0 5 0 2 acid AAA 244 98 98 125 1 0 2 Cysteine cys C 248 162 206 4 Aminobenzoic acid PABA 265 206 163 Homophenylalanine HPHE acid APA 198 258 286 198 138 112 0 5 0 4 Chloro phenylalanine Histamine HA 180 168 223 Glutamine GLN Q 84 187 84 112 8 10 Theanine THE 112 215 Bicine 290 260 2 4 Diamino n butyric acid DABA 203 142 245 Glycyl glycine dipeptide GLY GLY 117 144 201 Homocysteine HCYS 142 203 Methionine sulfone EZ faast User s Manual Table 2 continued ical Nam Abbreviatio bb Agi 973 Varia Methionine sulfoxide 229 182 138 S Carboxymethyl cysteine 144 203 262 Ornithine ORN 0 156 70 156 139 114 1 0 2 Glycyl proline dipeptide GPR G P 70 300 153 114 1 5 120 107 162 Lysine LYS K 170 128 153 170 128 1 0 2 Threonine aspartic acid dipeptide 218 360 130 Histidine HIS H 282 168 267 222 136 1 0 2 Naphthyl alanine Seleno cystine Se C C Hydroxylysine 2 isomers HLY OHLys 129 169 87 129 2
11. SPE tip or spilled sorbent particles will not affect the precision of the assay in any way 2 Add sample as follows For vapor phase hydrolysates Dry down any remaining acid in sample vial using a speed vac evaporator Pipette 100uL of Reagent 1 into sample vial to re dissolve amino acids Transfer sample if necessary to EZ faast sample vial and proceed For liquid phase hydrolysates pipette 100uL or less of the hydrolysate sample and 200uL of Reagent 2 into a vial keep the ratio of hydrolysate Reagent 2 1 2 and mix briefly The mixture should have a pH greater than 1 5 but less than pH 5 0 Check the pH of one sample with pH paper all other samples prepared by the same procedure should have a similar pH Pipette 25uL of mix and 100uL Reagent 1 into each sample preparation vial Note In both cases calculate the multiplication factor for quantitative analysis by taking into account the amount of sample and the volumes of HCI Reagent 2 or water used Note Amino acid standard mixtures come with the correct pH No pH adjustment is needed as described above Just add 100uL Reagent 1 to the amino acid standard mixture and proceed with the SPE as described at step 3 Figure 2 EZ faast User s Manual Gass Viar Line Up Figure 2 For each sample line up one glass sample preparation vial in the vial rack 3 Attach a sorbent tip to a 1 5mL syringe and loosen the syringe piston immerse the tip and pass
12. age and warranty information Please observe recom mendations for solvent bottle handling and syringe cleaning in Section 7 0 of this manual 2 4 Safety Although the concentration of all toxic components in any of the reagent bottles is low for safety reasons the sample preparation station should be placed in an exhaust hood and protective gloves and goggles should be worn When working with biological fluids please take any necessary precautions to prevent infection with blood borne pathogens Appropriate bio safety precautions and disposal of bio hazardous wastes should be followed EZ faast User s Manual 3 0 PREPARATION PROCEDURE 3 1 Setup The EZ faast kit packaging has been designed as an efficient workstation It holds a reagent tray a vial rack a pipette rack and a section for sorbent tips and vials To speed up sample preparation it is recommended that the workstation be arranged as shown in figure 1a By following directions and markings on the reagent box by breaking it along perfora tions it can be transformed into a reagent tray When the kit is not in use for several days the reagent tray figure 1b may be conveniently removed and placed in the refrigerator WorksTATION ARRANGEMENT FicunE 1 e To speed up sample preparation it is recommended that the workstation be arranged as shown below Figure 1a Figure 1b 3 2 Preparing the Eluting Medium The volume of prepared Eluting Medium dep
13. ame sample preparation vial see Figure 3 8 Pull back the piston of a 0 6mL syringe halfway up the barrel and attach the sorbent tip used in steps 3 6 EZ faast User s Manual THE SORBENT TIP IN THE VIAL FIGURE 3 Keep the sorbent tip in the sample preparation vial through steps 3 10 even while dispensing HPLC grade water at step 4 and eluting medium at step 7 9 Wet the sorbent with Eluting Medium watch as the liquid rises through the sorbent particles and stop when the liquid reaches the filter plug in the sorbent tip 10 Eject the liquid and sorbent particles out of the tip and into the sample preparation vial Repeat step 9 and 10 until the sorbent particles in the tip are expelled into the sample preparation vial Only the filter disk should remain in the empty tip see Figure 4 Discard the empty tip Keep the syringe as it can be reused with many other samples 11 Using the adjustable Drummond Dialamatic Microdispenser included transfer 50uL Reagent 4 into the sample preparation vial Caution Avoid cross contamination by not touching the inner wall of the sample vial with the tip of the Microdispenser The piston will ensure proper transfer of liquids into the vial without the need of touching the vial wall Use the same Microdispenser with both Reagents 4 and 5 There is no need to change Microdispenser tips between uses or to wash the dispenser between uses of Reagent 4 and 5 Warning Do
14. by vortexing for about 5 seconds Let the reaction proceed for 1 minute 15 Pipette DO NOT use the Microdispenser for the purpose 1001 Reagent 6 and re emulsify by vortexing for about 3 seconds The upper organic layer contains amino acid derivatives to be analyzed by gas chromatography on a ZB AAA column Gas Chromatographic Analysis Constant Flow Mode recommended GC FID NPD Settings Injection Split 1 15 250 C 2 0uL Carrier Gas Helium 1 5mL min Pressure Increase 3 kPa min alternative setting to constant flow Oven Program 32 C min from 110 to 320 C Detector 320 C Constant Pressure Mode GC FID NPD Settings Injection Split 1 15 250 C 2 0uL Carrier Gas Helium 8 psi 60 kPa or Hydrogen 30 kPa Oven Program 35 C min from 110 to 320 C Detector 320 C G EZ faast Kit Each kit includes a ZB AAA GC column or EZ faast LC column GC liners with GC kits sample prep and derivatization reagents sample prep vials AA standard mixtures SPE sorbent tips vial rack autosampler vials with inserts come with MS kits Microdis penser for Reagents 4 and 5 and demo video Description GC FID Free Physiological Amino Acid Analysis Kit GC MS Free Physiological Amino Acid Analysis Kit GC FID Protein Hydrolysate Kit GC MS Protein Hydrolysate Kit LC MS Free Physiological Amino Acid Analysis Kit with 250 x 2 0mm column LC MS Free Physiological Amino Acid Analysis Kit with 250 x 3 0mm column
15. ce the performance characteristics are not established Phenomenex products may be used in clinical diagnostic laboratory systems after the laboratory has validated their complete system as required by the Clinical Laboratory Improvements Amendments of 1988 CLIA 88 regulation in the U S or equivalent in other countries Trademarks EZ faast Sorbent Tips are patented Phenomenex Inc U S Patent 6 770 246 EZ faast is a trademark of Phenomenex Inc Phenex is a trademark of Phenomenex Inc FocusLiner and Auto Sep T are trademarks of SGE SoftGrip is a trademark of Hamilton Drummond is a registered trademark of the Drummond Corp Registered names trademarks etc used in this document even when not Specifically marked as such are not to be considered unprotected by Law 2005 2006 Phenomenex Inc All rights reserved EZ faast Kit Each kit includes a ZB AAA GC column or EZ faast AAA LC column GC liners with GC kits sample prep and derivatization reagents sample prep vials AA standard mixtures SPE sorbent tips vial rack autosampler vials with inserts come with MS kits Microdis penser for Reagents 4 and 5 and demo video Description GC FID Free Physiological Amino Acid Analysis Kit GC MS Free Physiological Amino Acid Analysis Kit GC FID Protein Hydrolysate Kit GC MS Protein Hydrolysate Kit LC MS Free Physiological Amino Acid Analysis Kit with 250 x 2 0mm column LC MS Free Physiological Amino A
16. cid Analysis Kit with 250 x 3 0mm column LC MS Protein Hydrolysate Kit with 250 x 2 0mm column LC MS Protein Hydrolysate Kit with 250 x 3 0mm column GC Free Physiological Amino Acid Standards SD1 SD2 amp 03 2mL vial x 2 GC Protein Hydrolysate Standard SD 2mL vial x 2 LC MS Free Physiological Amino Acid Standards for LC SD1 SD2 amp SD3 2mL vial x 2 LC MS Protein Hydrolysate Standard SD 2mL vial x 2 Order No KG0 7165 KG0 7166 KG0 7167 KG0 7168 KH0 7337 KH0 7338 KH0 7339 KH0 7340 60 7184 AGO 7263 ALO 7500 ALO 7501 Unit ea ea ea ea ea ea ea ea ea ea ea ea Phenex Vials This universal vial can be used in any autosampler that utilizes a 12 x 32mm vial It may be used in place of crimp top and snap ring top vials Eliminates the need of stocking many different style vials The top screws down in 1 3 turn and eliminates the chore of Crimping de crimping and snapping caps on Cap comes with a bonded in septa that eliminates septa slipping into vials Vials and caps with bonded in septa come in one convenient kit pack Description Clear wide mouth vial cap and septa kit pack with Rubber PTFE septa Silicone PTFE septa PTFE Silicone PTFE septa Amber wide mouth vial cap and septa kit pack with Rubber PTFE septa Silicone PTFE septa Clear wide mouth vial cap with pre slit septa Silicone PTFE septa SGE FocusLiners Description Thermo Electron 8000 series Carlo Erba S
17. d the first 3 5 injections when performing method calibration These injections act as primers and mask active sites inside the liner and chromatographic column Use subsequent duplicate runs for calibration Remember the SD vial should be placed in the freezer after use Allow standards to reach room temperature before use 4 9 Calibration of analytical results Calculations of amino acid levels in hydrolysed samples are performed by the Data Analysis portion of the software controlling the analytical instrument gas chromatograph Calculations and calibration are based on an internal standard Results are reported in the units entered for the internal standard and analyte levels in calibration mixtures Note nmols mL are equivalent to umols L FACCHI User s Manual e 4 10 Amino Acid and Protein Quantitation Calculations For additional information regarding protein quantitation calculations as well as example calculation spreadsheets please refer to reference CD included in kit 5 0 TROUBLESHOOTING Problem Decrease in peak height for some amino acids components of amino acid standard mixtures SD2 or SD3 Possibly related to improper sample storage See sample standard storage section 6 0 Decrease in peak height for basic amino acids Old eluting medium Prepare eluting medium daily based on the number of samples to be analyzed on that day Decrease in peak height or missing peaks for late eluting
18. eagent 2 into a glass vial and mix briefly If pH gt 1 5 pipette 25uL of mix and 100yL Reagent 1 into each sample preparation vial Section 3 4 2 3 Attach a sorbent tip to a 1 5mL syringe pass the solution in the sample preparation vial through the sorbent tip by slowly pulling back the syringe piston Pipette 200pL water into the sample preparation vial Slowly pass the solution through the sorbent tip and into the syringe barrel Detach the sorbent tip and discard the liquid accumulated in the syringe Pipette 200 Eluting Medium prepared fresh each day section 3 2 into the sample preparation vial Pull back the piston of a 0 6mL syringe halfway up the barrel and attach the sorbent tip Wet the sorbent with Eluting Medium stop when the liquid reaches the filter plug in the sorbent tip co c A Eject the liquid and sorbent out of the tip and into the sample preparation vial Repeat until all sorbent particles in the tip are expelled into the sample preparation vial Discard the empty tip 11 Using the Drummond Dialamatic Microdispenser transfer 50uL Reagent 4 12 Emulsify by repeatedly vortexing the solution for about 5 seconds Allow reaction to proceed for about 1 minute 13 Vortex the solution again for a few seconds to re emulsify the content of the vial Allow the reaction to proceed for at least one additional minute 14 Using the Microdispenser transfer 100pL Reagent 5 and re emulsify
19. ends upon the number of samples to be ana lyzed during the day 200uL sample The eluting medium should be prepared fresh each day 1 Use capped vials of appropriate size not included for preparation of the Eluting Medium 2 Combine 3 parts Reagent Eluting Medium Component 1 with 2 parts Reagent Eluting Medium Component Il in an appropriate sized vial see Table page 5 for reagent volumes based on number of samples Mix briefly 3 Store prepared eluting medium during the day at room temperature Discard any unused mixture at the end of the day Table 3 For your convenience check the table below to determine the volume of Eluting Medium components needed depending on your number of samples Number of Reagent 3A Eluting Reagent 3B Eluting Samples Medium Component I Medium Component 2 300uL 200uL 4 600 400pL 7 900uL 600pL 12 1 5mL 1 0mL 14 1 8mL 1 2mL 19 2 4mL 1 6mL 24 3 0mL 2 0mL 29 3 6mL 2 4mL 34 4 2mL 2 8mL 39 4 8mL 3 2mL 44 5 4 3 6mL 49 6 0mL 4 0mL EZ aast User s Manual 3 3 Protein Sample Hydrolysis 3 3 1 Background There are numerous published methods for protein hydrolysis all are compatible with analysis by the EZ faast procedure with minor modifications to the described method The most common methods use acid hydrolysis with 6M HCI in either a liquid or vapor phase Stein and Moore Methods in Enzymology 6 819 831 1963 Tarr et a
20. ic Analysis 10 IIroriblestootinge 13 Sample Storage and Stability 14 Cleaning and Care of Supplies 14 Quality ASSUFAnoe 14 E Ordering Information FACCHI User s Manual 1 0 Kir Components 1 1 Reagents Reagent Ingredients Volume Reagent 1 Norvaline 0 2 mM Internal Standard Solution N propanol 10 50mL Reagent 2 Sodium Carbonate Solution Na CO 90mL Reagent 3A Eluting Medium Component Sodium Hydroxide 60mL Reagent 3B Eluting Medium Component II N propanol 40mL Regent 4 Organic Solution Chloroform 4 vials 6mL each Reagent 5 Organic Solution II Iso octane 50mL Reagent 6 Acid Solution Hydrochloric Acid 1N 50mL SD Please refer to section 4 7 Amino Acid Standard Mixtures in the manual 2 vials of each SD 2mL each 1 2 Supplies Sorbent tips in racks ssseesessseseeeeeeererenennnnntntn tnter 4x96 Sample preparation Vial 4x100 Microdispenser 20 100uL Syringe 0 6mL 10 Syringe 1 5 sn 10 ZB AAA 10m x 0 25mm ID Amino Acid Analysis GC Column 1 FocusLiners 5 User EZ faast Demo Video and Reference CD sss 1 1 3 Materials Required but Not Supplied In Kit e 100pL 1mL pipette SoftGrip pipette Phenomenex P N 0 5968 or equivalent 30 300uL pipette SoftGrip
21. ingle Taper Gooseneck Liner Agilent Technologies Single Taper Gooseneck Liner PerkinElmer Single Taper Gooseneck Liner Shimadzu Single Taper Gooseneck Liner Varian Single Taper Gooseneck Liner Varian Double Taper Gooseneck Liner GC Model No 5880 5890 6890 Autosystem 17B 1075 77 1078 79 Dimensions ID Material Quartz IDxLxOD mm deactivated Wool Y N 5 x 105 x 8 0 B y Y 4 78 5 6 3 y Y 4x 92x 6 2 B y M 3 4x95x5 B y W 4 72 6 3 B y Y 3 4x 54x5 B y Y B Borosilicate Deactivated Yes y or No n Order No 4610 4613 4616 4619 0 4622 0 7507 Mfr P N 092046 092003 092095 092068 092025 092036 Unit 1000 pk 1000 pk 1000 pk 1000 pk 1000 pk 1000 pk Order No 0 4679 AGO 4680 AGO 4681 AGO 4683 AGO 4684 AG0 4685 Unit 5 pk 5 pk 5 pk 5 pk 5 pk 5 pk Detach Here 3882 14 FACCHI User s Manual EZ faast Amino Acid Analysis of Protein Hydrolysates by GC FID GC NPD Quick REFERENCE GuipE Summary of Procedure 1 For each sample line up one glass sample preparation vial in the vial rack 2 Vapor phase hydrolysate Dry down any remaining acid in sample vial using a speed vac evaporator Pipette 1001 of Reagent 1 into sample vial to re dissolve amino acids Section 3 4 2 Liquid phase hydrolysate Pipette 100uL sample hydrolysate and 200pL R
22. inish time of around 15 minutes A video included with this kit demonstrates the simplicity of the procedure Please be aware that some sample preparation steps described in the video may be different than what is described in this users manual Please use the video as a general guide but follow the exact Steps and sequence described in this manual 2 2 Amino Acids in Physiological and Protein Hydrolysate Samples The EZ faast method has been developed for the analysis of more than 60 aliphatic and aromatic amino acids including primary and secondary amines Further amino acids and related compounds may be analyzed with this kit A brief adjustment of gas chromatographic conditions may be necessary Please contact Phenomenex for method modifications and other LC and GC amino acid kits Table 1 Protein Amino Acids analyzed by the EZ faast Amino Acid Analysis Kit for Protein Hydrolysates by GC ical Abbreviation Alanine ALA A Glycine GLY G Valine VAL V Leucine LEU L Isoleucine ILE Threonine THR T Serine SER 5 Proline PRO P Asparagine ASN N Aspartic Acid ASP D Methionine MET M 4 Hydroxyproline HYP OHPro Glutamic Acid GLU E Phenylalanine PHE F Glutamine GLN Q Lysine LYS K Histidine HIS H Hydroxylysine 2 isomers HLY HYL OHLys Tyrosine TYR Y Tryptophan TRP Cystine C C Cys TRP is completely lost during acid hydrolysis use alternative h
23. ith sample preparation The amino acid standard mixture for protein hydrolysates SD is composed of the follow ing amino acids 200 nmoles mL each ALA GLU HLY LEU PHE THR VAL ASP GLY HYP LYS PRO TRP C C HIS ILE MET SER TYR TRP can be analyzed only in hydrolysates prepared under alkaline conditions 744 User s Manual Note the amino acids included in SD are the most widely analyzed for protein hydrolysates For assistance with additional amino acids in your hydrolysate sample please contact Phenomenex gt Figure 5 Norm E 704 60 4 504 401 5 304 2j 201 10 4 on 0 T T T T T T T T T 1 5 2 2 5 3 3 5 4 4 5 5 5 5 6 min 20 typical chromatogram of the amino acids standard solution included in this kit Column and instrumental settings as specified in section 4 1 4 2 4 8 Calibration Procedure Use the following standard amino acid mixtures for sample preparation and make dupli cate injections of each to generate the desired calibration Calibration Solution 1 25yL of SD solution plus 100yL Reagent 1 calibration level 50 nmoles mL ll 50uL SD solution plus 100 Reagent 1 calibration level Il 100 nmoles mL 11 100pL SD solution plus 100pL Reagent 1 calibration level Ill 200 nmoles mL The concentration of the internal standard IS in the sample prepared for chromatographic analysis is 200 nmoles mL Note Disregar
24. l In Microcharateriza tion of Proteins J E Shively ed Humana Press 1986 While these methods give good results for a majority of amino acids there are several amino acids that are either partially or completely destroyed by such methods and alternate hydrolysis methods must be used For convenience a common liquid and vapor phase method is shown hydrolysis reagents and supplies are not included with the EZ faast kit 3 3 2 Vapor Phase Hydrolysis The following is a sample method for vapor phase hydrolysis as a reference other meth ods may work better for your application 1 Transfer 1 20 nanomoles of protein into an autosampler vial insert 2 Lyophilize sample in a vacuum concentrator 3 hydrolysis vessel add 9891 6N constant boiling 10pL 5 Phenol and 1pL betamercaptoethanol 4 Add vial inserts into hydrolysis vessel and cap with minaret valve 5 Place vessel in an ice bucket and purge with nitrogen and vacuum several times and seal vessel under vacuum 6 Hydrolyze in oven at 110 C for 24 hours 7 Cool vessel and remove vial inserts 8 Dry down any remaining acid in sample vial using a speed vac evaporator Pipette 100uL of Reagent 1 into sample vial to re dissolve amino acids Section 3 4 2 9 Perform EZ faast procedure as per manual 3 3 3 Liquid Phase Hydrolysis The following is a sample method for liquid phase hydrolysis as a reference other methods may work better for your applicati
25. m 32 C min from 110 to 320 C Detector 320 C Constant Pressure Mode GC FID NPD Settings Injection Split 1 15 250 C 2 0uL Carrier Gas Helium 8 psi 60 kPa or Hydrogen 30 kPa Oven Program 35 from 110 to 320 C Detector 320 C When using a Shimadzu GC instrument please increase the injector temperature to 300 C For your convenience we have included the GC method for the Agilent 6890 GC system on the reference CD included with the kit To use this included method copy the method folder into the appropriate method folder in your software and load EZ faast User s Manual 4 3 Mode of operation For best resolution a rate of 35 C min is preferred with instruments operating in constant pressure mode only Electronic Pressure Control EPC or Advanced Flow Controller AFC equipped instruments should be operated preferably in constant flow or constant velocity mode With these instruments a temperature gradient of 30 32 C min is fast enough to elute the least volatile derivatives e g that of cystine with similar retention times to constant pressure mode If the instrument is not equipped with the EPC option you may use a pressure raise of 3kPa min 4 4 Liners Use the best deactivated liners supplied by the instrument manufacturer Good results were obtained with FocusLiners Phenomenex P N AGO 4680 fits Agilent and Varian 1177 injectors In general the liner should carry a plug of silani
26. not use regular pipettes and tips with Reagent 4 and 5 as they will contaminate the sample Use the included Microdispenser for Reagents 4 and 5 ONLY Note for all subsequent sample preparation steps use a vortex mixer set in the touch pulse mode to about 80 of max speed for any mixing operations 12 Emulsify the liquid in the vial by repeatedly vortexing for about 5 8 seconds During vortexing hold the sample vial firmly between fingers and keep it straight as you push it onto the vortex plate Do not let the vial wobble otherwise liquid may come out of the vial Allow reaction to proceed 1 minute or more The emulsion will gradually separate into two layers Note a longer reaction time than 1 minute each at steps 12 and 13 or later at step 14 does not affect results 13 Re emulsify the liquids in the vial by vortexing again for about 5 seconds Allow the reaction to proceed for one additional minute or more 14 Transfer with the Microdispenser 100uL Reagent 5 50uL twice for convenience and mix for about 5 seconds Let the reaction proceed for one more minute 15 Pipette DO NOT use the Microdispenser for this purpose 100uL Reagent 6 and vortex for about 5 seconds The emulsion will separate into two layers The upper organic layer contains amino acid derivatives to be analyzed by gas chromatography see GC set up and calibration in section 4 Sample this layer directly from the sample prep vial or use a Pasteur pipette to t
27. on Transfer 5 25 nanomoles of protein into a glass test tube 10x150mm Lyophilize sample in a vacuum concentrator Add 100 uL of 6N containing 4 Thioglycolic acid to tube Purge air from tube with vacuum and flame seal tube Hydrolyze in oven at 110 C for 22 hours Cool tube break seal and perform EZ faast procedure as per manual o 9 je ri 3 3 4 Limitations of Hydrolysis Methods While 6N acid hydrolysis is the most common procedure there are several limitations to this method ASN and GLN are deamidated to ASP and GLU and thus are quantitated as a FACCHI User s Manual mixture Peptide bonds of hydrophobic amino acids VAL ILE LEU may be difficult to break and require additional hydrolysis time up to 72 hours Residual oxygen in the hydrolysis vessel can increase the thermal breakdown of hydroxyl and sulfur containing amino acids typical recoveries for SER THR HYP and TYR range between 50 90 MET ranges from 25 75 Reducing hydrolysis time improves recoveries but reduces other amino acid yields see above GLY yields tend to exceed 100 especially for low level samples due to background protein contamination Finally both TRP and CYS are completely destroyed by acid hydrolysis and must be analyzed by alternate methods see below The above listed limitations are based on hydrolysis chemistry and are not related at all to the EZ faast process 3 3 5 Alternate Methods and References
28. ransfer part of it into an autosampler vial Figure 4 EZ aast User s Manual 3 5 Optimizing Sample Preparation Time For experienced users sample preparation proceeds in 7 8 minutes per sample This process can be further improved by preparing up to ten samples at a time For example at step 2 dis pense Reagent 1 and at later steps all other reagents in ten vials successively using the same pipette tip At step 9 after dispensing Reagent 4 vortex 2 3 vials simultaneously During each one minute wait at steps 10 12 prepare the next set of samples for SPE Tip Ficure 4 Wet the sorbent with Eluting Medium and stop before it gets to the filter then eject the liquid and sorbent particles out of the tip 4 0 Gas CuRoMATOGRAPHIC ANALYSIS 4 1 Column For EZ faast Amino Acid Analysis in Protein Hydrolysates by GC included The Zebron ZB AAA GC column for Protein Hydrolysate comes without a cage Connect the ends of the column in the usual manner rest the column coil on the oven bracket Keep the pieces of thermal thread spaced evenly around the column coil The maximum column temperature is 320 340 C Caution Always use safety glasses while installing the GC column 4 2 Instrument Conditions Constant Flow Mode recommended GC FID NPD Settings Injection Split 1 15 250 C 1 5 2uL with hot needle see section 4 6 Carrier Gas Helium 1 5mL min constant flow Oven progra
29. the solution in the sample preparation vial through the sorbent tip by SLOWLY pulling back the syringe piston in VERY small steps Caution Do not quickly pull back the piston Try to take at least one minute to pass the sample through the sorbent tip Watch as the liquid accumulates inside the syringe barrel and move the piston only as the accumulation slows down The syringe should be capable of drawing all sample and subsequent wash into the barrel If you run out of piston range detach the sorbent tip expel the solution from syringe barrel then reattach the sorbent tip and proceed with sample preparation Note the sorbent tip should stay in the sample preparation vial through steps 3 10 see Figure 3 even when dispensing reagents In case the sorbent tip cannot reach to the bottom of the vial tilt the vial to 45 push the tip into the vial gently and proceed with the SPE step 4 Pipette 200uL HPLC grade water into the same sample preparation vial 5 Pass the water through the sorbent tip and into the syringe barrel SLOWLY Drain the liquid from the sorbent bed by drawing air through the tip 6 Detach the syringe from the sorbent tip while keeping the tip inside the sample preparation vial Discard the liquid accumulated in the syringe Note save the syringe as it can be reused with many other samples For convenience place it into the pipette rack 7 Pipette 200uL Eluting Medium prepared fresh each day section 3 2 into the s
30. ydrolysis procedure to analyze for TRP ASN and GLN are quantitatively converted to ASP and GLU during acid hydrolysis EZ faast User s Manual Table 2 Comprehensive list of amino acids and related compounds analyzed by the EZ faast method as described in this manual Mass spectral information provided for confirmation when needed internal standard listed in bold GC MS lons Observed SIM L instrument Aallent 5973 Varian Saturn BLAN Ethanolamine 116 117 Alanine ALA A 130 88 130 70 1 0 1 Alliin 216 173 129 Sarcosine SAR 130 217 130 88 1 0 1 Glycine GLY G 116 207 116 102 2 0 1 a Aminobutyric acid ABA 144 102 144 102 1 0 2 Valine VAL V 158 116 116 98 158 0 6 0 2 Fluoro alanine B Alanine B ALA 129 158 98 B Aminoisobutyric acid B AiB 158 116 130 84 144 4 0 2 B Amino n butyric acid BABA 88 70 Norvaline NORV 158 72 158 116 Leucine LEU L 172 86 172 130 07 0 1 allo Isoleucine alLE 172 130 130 101 0 7 0 1 Isoleucine ILE 172 130 130 101 0 7 0 2 Homoserine HSER 102 128 143 Norleucine NLE 172 86 Threonine THR T 160 101 101 2 0 2 y Amino n butyric acid GABA 130 144 172 Serine SER S 146 203 101 86 2 0 2 Proline PRO P 156 243 156 114 1 0 1 Asparagine ASN N 155 69 113 2 215 3 Methyl cysteine 172 259 130 Pipecolic Acid HPRO Thioproline TPR 174 1
31. zed quartz or pesticide grade glass wool 4 5 Injection Split injection at a ratio of 1 10 to 1 20 is recommended e Injection volumes of 1 5 2 are optimal Quasi splitless injection mode will produce a 5 to 10 fold increase in sensitivity with some instruments In this mode the split valve should be closed for an initial 5 to 7 seconds Before selecting this injection mode it should be checked experimentally that no significant discrimination of late eluting amino acid derivatives takes place in comparison with common split injection Alternatively instruments equipped with EPC AFC can be operated at double initial head pressure for 6 10 seconds 4 6 Sampling Both autosampler and manual sampling can be performed If manual sampling is pre ferred hot needle injection is recommended to prevent discrimination of components with high boiling temperatures With this technique the sample plug is completely drawn into the syringe barrel leaving the needle empty The needle is inserted and kept in the hot injector for about two seconds before injection 4 7 Calibration Standards For quantitation purposes mixtures of amino acid standards should be prepared following the Sample Preparation by SPE and Derivatization procedure described in this manual in Section 3 4 Amino acid standard mixtures should be stored in the freezer With standards pH adjustment is necessary Simply add 100pL Reagent 1 Internal Standard solution and proceed w

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