1

ก����� Performance verification ��� HPLC ������� ������� ��!"

!�� ��!�# : Performance verification, HPLC High performance liquid chromatography (HPLC) ����� !�"!ก$%&"�!%$'() *+�%'ก,-./&0ก$%10ก12'ก$%&"�!%$'()�%"3$4 �5$3$67/&"�!%$'()�%"3$46�./$�89$: ; �79� 0$ ,$($% <$%�!3* �!%=+,:<5$,$: 12'<"+:1&.2/,3 �>=+,6(/?2ก$%&"�!%$'()���� *+�9$�7=+,@=,A'8/,: 5$6(/%'-- HPLC 5$:$�B./,09$:3*�%'<" C"D$> AE:8/,:3*ก$% &�<,-ก$% 5$:$�F.0ก$% .<,- GH:%'-- (Holistic test) 12'ก$% .<,-1892'3,.J2 (Modular test) 6�79&:%'0'�&2$ *+�(3$'<3 ก$% .<,- GH:%'--<$3$%@�5$&"C*ก$%&"�!%$'() *+ 5$,0J93$67/�>=+,�%'�3"�?2L,:%'--&9$?2ก$%&"�!%$'() *+B./0G:����B�8$3!&$38/,:ก$%(%=,B39 ����ก$%�5$�,$&"C* .<,-6�79&:ก$% 5$ Performance qualification 3$67/ <9&�ก$% .<,-1892'3,.J2A'67/�ก4M)ก$% .<,- *+ 5$6�<9&� Operation qualification

6�- !&$3�*HA'<%O��P>$'ก$% 5$ Performance verification กG-%'-- HPLC *+67/ UV-Visible detector � 9$�GH� F.0ก$% &�<,-12'!&$3@*+<%O�6�8$%$: *+ 1 <5$(%G- Detector 7�".,=+� ; ,$A3*ก$% &�<,-�P>$'3,.J2 Detector *+,$A18ก89$:กG�B�-/$:

%�����&' 1 Performance Attributes for HPLC Modules and Test Frequency

��456 !78 �9�%: ;ก8<"ก����4 !=���&'

Pump Flowrate accuracy ± 2 % of the set flowrate 6 months Gradient accuracy ± 1 % of the step gradient composition 6 months Pressure test Proper functioning check valve

Pressure decay: < 75 psi/min No leak from the pump

6 months

Injector Precision 1% RSD 6 months Linearity r ≥ 0.999 12 months

Carryover < 1 % 6 months Detector Wavelength accuracy ± 2 nm 6 months Linearity of response r ≥ 0.999 12 months

Noise and drift Noise: 10-5 AU Drift: 10-4 AU/h

12 months

Column compartment

Temperature accuracy ± 2 °C of the set temperature 6 months

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ก����� Performance verification 1. Pump

1.1 Flowrate accuracy !&$3@Jก8/,:L,: Flowrate A'3*?289,!&$3@Jก8/,:L,: Retention time 12' Resolution &"C*�p"-G8" F.0�%G- Flowrate L,: Pump qE+:,$A 5$6(/3* Back pressure 12/&�กs-%&-%&3 mobile phase ./&0L&.&G.�%"3$8%>%/,3กG-AG-�&2$A�@E:L*.�%"3$8% !5$�&�!&$3@Jก8/,:L,: flowrate 8$3�ก4M)6�8$%$: *+ 1 1.2 Gradient accuracy !&$3@Jก8/,:L,:ก$%&"�!%$'()./&0%'-- Gradient LEH�กG-!&$3@Jก8/,:L,:ก$%��2*+0�1�2:<9&��%'ก,-L,: mobile phase 6�79&:�&2$L,:ก$%10ก &"C*�p"-G8" ��=+,:A$ก%'-- Gradient 3* 2 1-- B./1ก9

- High pressure gradient qE+:A'67/�tu3 1 8G&89,8G& 5$2'2$0 1 7�". F.0 G+&B�A'67/�tu3 2 8G&����%'-- Binary F.0ก5$(�.6(/���� Channel A กG- B

- Low pressure gradient A'67/�tu38G&�.*0&18967/ Proportional valve ?<38G& 5$2'2$0 F.0 G+&B�A' 5$B./ 4 7�".(%=,%'-- Quarternary ก5$(�.���� Channel A, B, C 12' D

ก$% &�<,-!&$3@Jก8/,:L,:%'-- gradient 5$B./F.0ก$%8".8$3!9$1,-q,%)1-�q) *+��2*+0�1�2:8$3,G8%$<9&�L,:8G& 5$2'2$0L,:1892'!J9 Channel กG-�&2$ *+.5$��"�B� <5$(%G-ก%4*%'-- Quarternary A' 5$ก$% &�<,- Channel A กG- B 12' Channel C กG- D ก$%8".8$3ก$%��2*+0�1�2:,G8%$<9&�L,:8G& 5$2'2$0A'67/<$% *+6(/!9$1,-q,%)1-�q)6<92:6�8G& 5$2'2$0 �79� ,'q*F8� 8G&,09$:F�%1ก%3 Gradient 1<.:6�8$%$: *+ 2 �3=+,>2,8!9$1,-q,%)1-�q)กG-�&2$A'B./ก%$w6�D$> *+ 1

%�����&' 2 AB�Cก�� �����9ก���=� �9!=���5ก%D������E99 Gradient

Step Time (min) Flow rate (mL) Solvent Channel A (Methanol)

Solvent Channel B (0.5 % acetone in methanol)

1 0 x 1 3 100 0 2 1 x 2 3 0 100 3 2 x 3 3 20 80 4 3 x 4 3 40 60 5 4 x 5 3 60 40

6 5 x 6 3 80 20 7 6 x 7 3 100 0 8 7 x 8 3 100 0

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ก$%&"�!%$'()!&$3@Jก8/,: 5$F.0&G.!9$1,-q,%)1-�q) *+ 100 % Channel B ���� !9$ H *+!9$1,-q,%)1-�q) B1, B2,B3 12' B4 !&%A'���� 80%, 60%, 40% 12' 20% L,:Channel B qE+: *+Baseline A'���� 0 % L,:Channel B 12'!9$1,-

P���&' 1 ก��!���=� Gradient accuracy Q�กก��R q,%)1-�q) *+B./�3=+,�5$B�>2,8 กG-<9&��%'ก,-L,: Mobile phase *+B./8$3 {|p*A'B./!9$ Linearity L,:%'-- Gradient

1.3 Pressure test ����ก$%8%&A<,-ก$%%G+&L,:%'-- qE+:A'3*?289, Flowrate accuracy %&3@E:ก$%10ก12'ก$%&"�!%$'() &"C* .<,- .5$��"�ก$% Prime �tu3./&0�3 $�,2A�1�96A&9$B393*w,:,$ก$}6�%'-- �~. 9, $:,,กL,:�tu3./&0 Fitting 7�". Dead-nut 12'8GH:!9$ Pressure limit *+ 4000 �,�.)89,8$%$:�"H& �%G- Flowrate *+ 1 3"22"2"8%89,�$ * L4' *+!&$3.G��>"+3LEH��%'3$4 3000 �,�.)89,8$%$:�"H& 2. Flowrate 2:3$ *+ 0.1 3"22"2"8%89,�$ * -G� Eกก$%��2*+0�1�2:!&$3.G� �5$3$>2,8ก%$wA'3*2Gก|4'8$3D$> *+ 2 1<.:�t�($ก$%%G+& *+,$A3*B./

6�D$> *+ 2 6�ก%4* *+�%G- Flowrate A$ก 1 3"22"2"8%89,�$ *B����� 0.1 3"22"2"8%89,�$ * !&$3.G�A'!9,0;LEH�B�A�@E: 4000 �,�.)89,8$%$:�"H& 189@/$!&$3.G�B39LEH�1891ก&9:LEH�2:A'1<.:&9$3*ก$%%G+&3$ก *+ Check valve 189@/$!&$3.G�<$3$%@LEH�B�A�@E:4000 �,�.)89,8$%$:�"H& �tu3A'(0O.ก$% 5$:$�,G8F�3G8" @/$�tu3 5$:$��ก8"!&$3.G�!&%A'2.2:B39�ก"� 10%

P���&' 2 S6���ก����� Pressure test 6� 5 �$ *(%=,� *0-�ก4M)6�8$%$: *+ 1 189@/$3*ก$%2.2:�%s&3$ก�9$A'3*�t�($ *+ Pump seal (%=, Check valve

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2. Injector

2.1 Precision ก$% &�<,-!&$3� *+0:L,: Injector F.0ก$%P*.<$%2'2$03$8%�$�(%=,<$%2'2$0

8G&,09$: ,09$:�/,0 6 qH5$12/&!5$�&� % RSD L,:>=H� *+>*! 2.1 Precision ก$% &�<,-!&$3� *+0:L,: Injector F.0ก$%P*.<$%2'2$03$8%�$�(%=,<$%2'2$0

8G&,09$: ,09$:�/,0 6 qH5$12/&!5$�&� % RSD L,:>=H� *+>*! 2.2 Linearity ����ก$% &�<,-ก$% 5$:$�L,: Injector *+<$3$%@��2*+0��%"3$8%L,:ก$%P*.

<$%2'2$0B./ F.0 G+&B� Autosampler <$3$%@ 5$B./ F.08/,:8%&A<,-กG-�%"3$8%L,: Sample loop �79� Sample loop 3*�%"3$8% 100 B3F!%2"8% ก$% &�<,-A'P*.<$%2'2$03$8%�$�(%=,<$%2'2$08G&,09$: *+ 5, 10, 20 12' 100 B3F!%2"8% A$ก�GH��5$!9$1,-q,%)1-�q) *+B./>2,8กG-�%"3$8%12/&8%&A<,-!9$ Correlation coefficient

2.3 Carryover ����ก$% &�<,-ก$% 5$:$�L,: Injector F.0ก$%P*.<$%2'2$03$8%�$�(%=,<$%2'2$08G&,09$: *+3*!&$3�L/3L/�<J: *+<O.6�ก$%&"�!%$'()12/&P*.<$%2'2$0 Blank ก$% Carryover 8/,:B39�ก"� 1 % 3. UV-Visible detector

3.1 Wavelength accuracy !&$3@Jก8/,:L,:!&$30$&!2=+�L,: Detector ,$A�ก".A$กก$% 5$:$� *+?".>2$.L,: Monochromator qE+:3*?289,!&$3B&L,:ก$%&"�!%$'() ก$% &�<,- 5$B./F.067/<$%2'2$0 *+ %$-!9$ก$%.J.ก2=�!2=+�1<: �79� <$%2'2$0 Erbium perchlorate hexahydrate2 P*.�L/$B� *+ Flow cell 12/&�5$!9$1,-q,%)1-�q)3$>2,8กG-!&$30$&!2=+� 3.2 Linearity of response ����ก$% &�<,- Linearity L,: Detector 5$B./F.0ก$%67/<$%2'2$03$8%�$�P*.�L/$<J9 Flow cell !&%A'67/!&$3�L/3L/��%"+3 *+ 0 A�@E:!&$3�L/3L/� *+6(/!9$1,-q,%)1-�q)3$กก&9$ 1.5 �>%$'79&:�*H����ก$%8%&A<,- Detector ,$A3*ก$%�-*+0:�-�L,: Linearity A$ก?2L,: Stray light 3.3 Noise and drift <$�(8OL,: Noise 12'ก$% Drift �ก".B./A$ก %'--,"�2sก %,�"ก<) �tu3 Intensity L,: Lamp !&$3<ก�%กL,: Flow cell 12',O4(DJ3" *+B39!: *+ 3*?289,!&$3B&L,:ก$%&"�!%$'() ก$% &�<,- 5$B./F.0ก$% Run �3 $�,2 Noise *+�ก".LEH�3* 2 1-- Short term noise A'3*?289,ก$%&"�!%$'()�%"3$4qE+:A'>-6�ก$%&"�!%$'() *+!&$3�L/3L/�8+5$; <9&� Long term noise 3GกA'>-6�<9&�>*ก *+,,ก3$ *+ Retention time 3$ก; %J� *+ 3 Short term noise A'-G� EกF!%3$F 1ก%36�79&: 15 �$ * 2$ก�</�L�$�-�12'29$:L,:>*ก12/&1-9:,,ก���� 15

5

<9&� 1892'<9&�� 9$กG- 1 �$ * !&$3<J:L,: Noise 1892'<9&�!=, Y �5$�,$!9$ Y %&3กG�12/&($%./&0 15 <9&� A'B./!9$1,-q,%)1-�q)qE+:����!9$ Short term noise

D$> *+ 4 1<.: Long term noise <$3$%@!5$�&�B./A$กF!%3$F 1ก%36�D$> *+ 3 B./ F.0ก$%2$ก�</�L�$�6(/ !%,-!2O3AO.ก2$: X F.01-9:���� 2 79&: 79&:2' 10 �$ * A$ก�GH�6(/&G.!&$3<J:L,:�</�L�$� Z @/$79&:B(�3*!9$3$ก ก&9$6(/ก5$(�.����!9$ Long term noise

P���&' 3 ก��!���=�!Z� Short term noise D$> *+ 5 1<.:ก$%!5$�&�!9$ Drift L,: %'--F.0ก$% Run 12'-G� EกF!%3$F 1ก%3 �����&2$ 1 7G+&F3: A$ก�GH�2$ก�</�L�$� GH:<9&�-�12'29$:L,: Noise 2$ก�</� Baseline 82,.79&:12/&!5$�&�!9$1,-q,%)1-�q) *+<J: *+<O. A'����!9$ Drift

P���&' 4 ก��!���=�!Z� Long term noise 4. Column compartment (%=,<9&� Column oven !&$3@Jก8/,:L,:ก$%!&-!O3,O4(DJ3"A'3*?289,ก$%10ก qE+:,$A 5$B./F.067/ *+<,-� *0-12/&&$:6�<9&� Column oven 12/&,9$�!9$,O4(DJ3" *+��2*+0�1�2:

P���&' 5 ก��!���=�!Z� Drift ����E99

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;�ก ���D���:�

1. Chung Chow Chan, Herman Lam, Y. C. Lee and Xue-Ming Zhang (Eds.). Analytical Method

Validation and Instrument Performance Verification. New Jersey: John Wiley & Sons, Inc., 2004, Chapter 11.

2. American Society for Testing and Materials. Standard Practice for Testing Variable Wavelength Photometric Detectors Used in Liquid Chromatography. Annual Book of ASTM Standards: E 1657-98. Washington. DC.: ASTM, 2006.

F!%:ก$%�!3* ก%3&" 0$}$<8%)-%"ก$% F %. 0 2201 7232 E-mail: nart@dss.go.th ก%กp$!3 2553