1. 9100 HPLC Trouble shooting

2. Liquid Chromatography
1. Solvent
4. Injector
2. Degasser
3. Pump
5. Column
6. Detector
7. PC & SW

3. 1. Solvent What to check? ① What is the purity of Mobile phase?
-> Organic solvent : HPLC grade
Water : 18.2 mΩ/cm2 (Ultra pure grade)
② How about miscibility?
Difference value of Miscibility between two solvents should be less than 5.
(Refer to Miscibility Index at next page.)
③ Do you perform Filtration / Sonication sufficiently?
④ Enough quantity of Mobile phase in solvent reservoir?

4. Solvent miscibility Acetone 15 Acetonitrile 11 n-Butyl acetate 22
N-Butyl alcohol
Chlorobenzene 21
Chloroform 19
Cyclohexane 28
Dichloromethane 20
n,n-Dimethylformaide 12
Dimethyl sulfoxide 9
1,4-Dioxane 17
Ethyl acetate
Ethyl ether 23
Heptane 29
Hexane
iso-Octane
Isobutyl alcohol
Isopropyl alcohol
Methanol
Methyl ethyl ketone
Pyridine 16
Tetrahydrofuran
Toluene

5. Probable cause & Remedy
① Air-bubbles in mobile phase - When using immiscible solvents -> Sonication is necessary and use Premix solvent in one solvent reservoir. - Cleaning Slip on filter ② Unstable Baseline / Solvent peak increased - Check Water status. -> Use a transparent bottle to see it and replace it everyday.
Remove slip on filter from the tubing
and perform sonication with Water
for 30min and Methanol for 30min.

6. 2. Degasser What to check? ① Is LED of Power, Vacuum On?
- It generates the motor vibration with noise.
② Is Slip on filter in Mobile phase?
③ Are all tubes filled with Mobile phase?

7. Probable cause & Remedy
① Air bubble is in Mobile phase
- Check whether Vacuum LED is On, and vibration from Degasser.
-> If vacuum doesn`t generate, check Chamber leakage, Motor failure, or Mainboard failure.
- Check Solvent miscibility-> Refer to Miscibility Index.
② R.T is slightly shifted.
- Check if all tubings are filled with Mobile phase.
-> Perform prime purge again,
-> Put all in a same solvent reservoir when using one channel.
-> When HPLC is Ready to operate? = All tubing is filled with Mobile phase.

8. 3. Pump What to check? ① Is LED of Power, Connect On?
② Are all tubes and fittings connected properly?
③ Is there any leakage?
④ Do you replace Rinsing reagent with fresh one?
-> When using Buffer solution (salt) in mobile phase, fill the rinsing bottle with 100% of Water. If not, fill it with 50% of Methanol.  Replace it Everyday
⑤ Do you perform Prime Purge to all channels from software?
-> Perform all channels at least 2min each before running the Pump.

9. Probable cause & Remedy
Quarternary Pump
Prime Purge valve
Inline Filter
Four Check valves
Plunger
High pressure seal
Purge Vent line
Gradient Mixer
Rinsing
bottle
To Waste
To Injector

10. ① When Pressure is not applied; -> Perform Prime Purge again
① When Pressure is not applied; -> Perform Prime Purge again. - Purge valve position Clock wise turn: Moves to Injector, Counter clock wise turn: Moves to waste -> Remove the tubing connected to inlet of column and check that mobile phase comes out from the tubing. If it comes out, reduce the flow rate and connect the tubing to column again. -> Check the vibration generates from pump head by finger. - If Pump doesn't generate the vibration, check stepping motor, power supply from main board and power board.

11. ② Pressure is fluctuated higher than 30psi
② Pressure is fluctuated higher than 30psi. -> Perform Prime Purge again. -> Clean Inline filter: Sonicate it with Water for 30min and Methanol for 30min and re-install it. -> Clean four Check Valves : Sonicate them with Water for 30min, 20% of Nitric acid for 3min, and Water for 30min again and re install them. ③ Pressure drops to half from the expected value. ->Clean Check valve or Replace.

12. ④ Increase of solvent volume in the Rinsing bottle -> Check High pressure seal leakage. ⑤ Peak shape is suddenly broaden or RT is shifted -> Mixing Valve doesn’t open or close as per solvent ratio setting. -> Check if same pressure is applied for A,B,C, and D line with same solvent. -> Mixer replacement ⑥ Scratch on Plunger -> It reduces life time of high pressure seal. Replace it after 3times of seal replacement.

13. 4. Injector What to check? ① Manual Injector (Rheodyne valve injector)
- Do you inject the sample to Load position?
- Do you use LC syringe?: GC syringe is not compatible.
- Do you clean the syringe sufficiently before injection?
- Is injection volume three times higher than loop volume?
Loop volume 20 uL => Inject 60 uL
② Autosampler (Midas, Alias, Optimas)
- Is the washing bottle filled with washing solvent?
- Is washing solvent miscible with mobile phase?
- Do you perform the manual wash procedure before start?

14. Probable cause & Remedy
Sample loop :
Check loop volume.
Stator : Check the joints.
Rotor seal : Flow path is worn out and solvent leakage occurs at front or back side.

15. Manual Injector (Rheodyne valve injector)
① Leakage occurs at front side after the syringe remove
 Check the wear of flow path on rotor seal.
Rotor seal replacement
② Leakage occurs at the joint connected to column
 Column or tubing is clogged by some contamination and pressure is applied higher than 4000psi.
Replace or clean the column and tubing.
③ Software doesn`t start when it turns to inject position.
 Check Remote cable and main board.

16. Autosampler (Alias, Midas, Optimas)
① Reproducibility of peak area is poor or peak doesn`t appear.
-> Check the washing solvent.
-> Remove air-bubble in the
syringe.
-> Check sample quantity in
the vial.
(500 uL at least. If less than
500uL, use an insert.)
Washing
bottle
Syringe
Needle
Sample tray

17. ② Ghost peak appears at RT of solvent peak
② Ghost peak appears at RT of solvent peak./Air-bubbles generates in the syringe continually.
-> Check miscibility between washing solvent and mobile phase.
--> Use Same solvent for washing and mobile phase.
-> Use only Water as washing solvent if mobile phase contains high concentration of buffer (salt).
③ Needle damaged
-> Check the alignment and replace the needle.

18. 5. Column What to check? & C.C
① Is Sample diluted completely and clean?
 Sample preparation is necessary
such as Filtering and Sonication.
 Use a guard column.
② Be aware of column information.
 Consider PH range, flow rate, mobile phase limitation.
③ Perform column conditioning and cleaning?
 Clean the column with 100% of Water after using buffer.
④ Is Suitable temperature used?
 General C18 column is used between 25 ~ 55℃.
 Check the column specification.

19. Pre-heat mobile phase temperature before it moves to the column.
Probable cause & Remedy
① Poor peak area reproducibility
Check the leakage.
② Poor RT reproducibility
Check column oven temp.
Use Pre-Heating Line.
③ High pressure
Increase the temp. within column spec.
Use reversed column direction
Pre-heating line :
Pre-heat mobile phase temperature before it moves to the column.

20. 6. Detector What to check? ① UVD/PDA(9120, 9160)
- Is Ready/Run LED flickering?
 When Ready/Run is flickering, UVD/PDA can connect to software.
Ref/Sam energy is around 1:1 after flowing mobile phase?
 If Ref is less than 50 nA, replace D2 lamp with new one.
② RID
Stabilization time is enough?
 Few hours is necessary after temp. reaches to setting point.
Lab temperature is higher than 15 ℃?
 RID makes an alarm when lower than 15 ℃ .

21. ③ ELSD
- Is quantity of Nebulizing Gas enough?
 It consumes 1L per minute, and one cylinder can not be used for one day in general.
- Is mobile phase B.P lower than sample B.P?
 If sample B.P lower, all sample is volatilized and peak doesn`t appear on chromatogram.
 Volatile buffer can be used such as Formic, Acetic, TEA, TFA.

22. Probable cause & Remedy
Common problem
① Steady and continuous pulse appears
 When using immiscible solvents,
: Use miscible solvents or premix them in a same bottle.
When using 90:10 solvent ratio,
: Premix them in a same bottle.
Use one solvent bottle with RID.
Unstable Column pressure / temperature.
: Clean pump and column
/Use column oven and pre heating line.

23. ② Noise level increased
 Check air-bubbles and solvent status.
: Miscibility / Degassing / Prime purge
 UVD/PDA : Check UV-Cutoff of mobile phase.
 RID : Noise level is various depending on RI of mobile phase.
 ELSD : Gas pressure is lower or nebulization is unstable.

24. ③ No sample peak
- Solvent peak appears but sample peak is not observed.
 Sample concentration: Check LOD of detector.
 Injection volume: Concentration is Ok, but injection volume is not enough.
④ No solvent and sample peak
 Baseline is observed only with pulse and noise.
: Sample injection error. Check injector and syringe.
 Flat baseline: Communication is lost.

25. UVD/PDA(9120, 9160)
W Lamp
9120
Flow Cell
Leakage sensor
D2 Lamp
9160

26. ① Solvent peak is increased or ghost peak appears.
 Mobile phase is contaminated or purity is poor.
: Check the purity and replace Water everyday.
② Injection vol. and conc. are same, but peak area is reduced.
 Check Ref energy and replace the lamp if necessary.
③ Injection vol. and conc. are same, but peak doesn't appear.
 Air-bubbles are in Flow cell.
: Flow mobile phase at 2~3ml/min without column or apply back pressure by finger until sample energy is increased.

27. RID(750F, 9170)
① Continuous beep sound after turning on
- Check room temperature: RID works higher than 15C.
② Negative peak at peak tail or Spike peak observed
- Purge valve leakage : Place solvent reservoir higher than RID and use wide ID of outlet. -> Purge valve replacement
③ All peaks are negative.
- Change Polarity setting.
④ Optical balance is not within ± 20% and signal value is high.
- Adjust mirror position with two hex screws at rear side of optic.

28. ELSD(Zam3000, 9180 series)
① After purging it with Gas for 10minuntes and connect mobile phase. After use, stop flow rate and purge it with Gas for 10min then turn it off.
② RT and intensity could be shifted when Gas cylinder is replaced.
9160

29. 7. PC & SW What to check? \\\ ① YL9100 - IP setting on PC is correct?
- LAN Hub works properly?
- PC LAN card works properly?

30. Probable cause & Remedy
① YL- clarity shows Not Connect message.
- Re-boot PC and Device and add device drivers again on
configuration window. (Refer to user manual for details.)
- Check MPU board. (communication board)

31. 8. etc. What to check? Error LED for YL9100 LC
① Solvent leakage: Open front cover and dry the leakage sensor completely.
② MPU Board failure : Hub and LAN is working properly but device doesn`t communicate to software.
③ Lamp ignition failure for UVD/PDA : Lamp or Power board replacement.
④ High pressure error for Pump: Check the pressure limitation.
Check purge valve position during prime/purge.