Difference between revisions of "Measurement of Sugars and Alcohol on HPLC"
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== Materials and Equipment ==<!-- Any tubes, pipette tips ect. --> | == Materials and Equipment ==<!-- Any tubes, pipette tips ect. --> | ||
| − | + | * HPLC withRefractive Index Detector | |
| − | + | ** Bio-Rad Aminex HPX-87H Column | |
| − | + | ** Cation H deashing gaurd column (BioRad #125-0129) placed inside the column heating unit, | |
| − | + | * 2 µm filters for syringe end | |
| + | * 1 ml syringe | ||
| + | * HPLC vials | ||
| + | * 1ml Pipette with pipette tips | ||
==Reagents==<!-- Test kits go here if used--> | ==Reagents==<!-- Test kits go here if used--> | ||
| Line 21: | Line 24: | ||
! CAS Number | ! CAS Number | ||
|- | |- | ||
| − | | <!-- Common Name--> | + | | Sulfuric Acid Mobile Phase<!-- Common Name--> |
| − | | | + | |0.005 |
| − | | <!-- By weight or volume? g/L, Mol, ect..--> | + | | <!-- By weight or volume? g/L, Mol, ect..-->Molar |
| − | | <!-- Universal Identifier, regardless of manufacturer --> | + | | 7664-93-9<!-- Universal Identifier, regardless of manufacturer --> |
|} | |} | ||
| Line 30: | Line 33: | ||
=== HPLC Settings === | === HPLC Settings === | ||
| − | Use the following settings for HPLC: Column: | + | Use the following settings for HPLC: Column: Injection volume: 50 µL, Flow rate: 0.6 ml min<sup>-1</sup>, Column temperature: 65°C, Detector temperature: 30°C, Run time: 30 minutes. HPLC should be calibrated at 4, 2, 1, 0.5, and 0.25 g L<sup>-1</sup> with glucose. |
| − | |||
| − | |||
=== Sample Preparation: === | === Sample Preparation: === | ||
Solids should be removed to the extent possible with centrifugation and filtration. Samples should be able to pass through a 2um syringe without significant resistance. | Solids should be removed to the extent possible with centrifugation and filtration. Samples should be able to pass through a 2um syringe without significant resistance. | ||
| Line 38: | Line 39: | ||
1. Centrifuge sample for 5min at 5000 rcf in centrifuge or microcentrifuge. | 1. Centrifuge sample for 5min at 5000 rcf in centrifuge or microcentrifuge. | ||
| − | 2. Pipette ~1.25 ml of decant into a 1.5ml microfuge tube taking care to avoid transferring solids. Place microfuge tubes in Microcentrifuge and centrifuge for 10min @ 16000 rcf. | + | 2. Pipette ~1.25 ml of decant into a 1.5ml microfuge tube taking care to avoid transferring solids. Place microfuge tubes in Microcentrifuge and centrifuge for 10min@ 16000 rcf. |
3. Pull a sample with a 1ml syringe from the microfuge tube and filter though a 0.2 µm filter into the HPLC vial. | 3. Pull a sample with a 1ml syringe from the microfuge tube and filter though a 0.2 µm filter into the HPLC vial. | ||
| Line 55: | Line 56: | ||
7. Process HPLC results | 7. Process HPLC results | ||
| + | |||
| + | ==Common Mistakes== | ||
| + | |||
| + | {| class="wikitable sortable" | ||
| + | |- | ||
| + | ! Mistake | ||
| + | ! Evidence of the Mistake | ||
| + | !Cause | ||
| + | ! Effect on Results | ||
| + | |- | ||
| + | | | ||
| + | | <!-- (How can you tell if the mistake has been made?)(How can you tell if the mistake has been made?) --> | ||
| + | |<!-- What is fundametnally happening | ||
| + | --> | ||
| + | | <!-- (Higher, lower, flatline?) --> | ||
| + | |} | ||
| + | |||
| + | ==Precision and Interference== | ||
Latest revision as of 03:21, 1 March 2019
Contents
Authors:
This method is also called:
This method is adapted from:
Discussion
Materials and Equipment
- HPLC withRefractive Index Detector
- Bio-Rad Aminex HPX-87H Column
- Cation H deashing gaurd column (BioRad #125-0129) placed inside the column heating unit,
- 2 µm filters for syringe end
- 1 ml syringe
- HPLC vials
- 1ml Pipette with pipette tips
Reagents
| Reagent | Concentration | Units | CAS Number |
|---|---|---|---|
| Sulfuric Acid Mobile Phase | 0.005 | Molar | 7664-93-9 |
Procedure
HPLC Settings
Use the following settings for HPLC: Column: Injection volume: 50 µL, Flow rate: 0.6 ml min-1, Column temperature: 65°C, Detector temperature: 30°C, Run time: 30 minutes. HPLC should be calibrated at 4, 2, 1, 0.5, and 0.25 g L-1 with glucose.
Sample Preparation:
Solids should be removed to the extent possible with centrifugation and filtration. Samples should be able to pass through a 2um syringe without significant resistance.
1. Centrifuge sample for 5min at 5000 rcf in centrifuge or microcentrifuge.
2. Pipette ~1.25 ml of decant into a 1.5ml microfuge tube taking care to avoid transferring solids. Place microfuge tubes in Microcentrifuge and centrifuge for 10min@ 16000 rcf.
3. Pull a sample with a 1ml syringe from the microfuge tube and filter though a 0.2 µm filter into the HPLC vial.
4. Run through HPLC using startup Wizard (The following is a generalized procedure for Waters Software)
a. Select LC
b. Select number of injections (depends on what is being analyzed)
c. Injection volume is 50 (micro liters) and run time is 35 minutes, unless stated otherwise.
d. Select Method and name samples
6. Fill out the name of the HPLC run in the notebook
7. Process HPLC results
Common Mistakes
| Mistake | Evidence of the Mistake | Cause | Effect on Results |
|---|---|---|---|
