HPLC设备的确认(中英)QualificationofHPLCEquipment解读

European Directorate for the Quality of Medicines & Healthcare
OMCL Network of the Council of Europe QUALITY MANAGEMENT DOCUMENT
欧洲委员会OMCL 网络
质量管理文件
PA/PH/OMCL (11) 04
QUALIFICATION OF EQUIPMENT
设备的确认
ANNEX 1: QUALIFICATION OF HPLC EQUIPMENT
附件1: HPLC (高效液相色谱仪)设备的确认
COUNCIL CONS EIL OF EUROPE
DE UEUROPE
ANNEX 1 OF THE OMCL NETWORK GUIDELINE
“ QUALIFICATION OF EQUIPMENT'
OMCL网络指南“设备的确认”之附件1
QUALIFICATION OF HPLC EQUIPMENT
HPLC设备的确认
Introduction 概述
The prese nt docume nt is the first Annex of the core docume nt “ Qualificati on of Equipme nt
should be used in comb in ati on with it whe n pla nning, perform ing and docume nting the HPLC equipme nt qualificati on process.
本文件是核心文件“设备的确认”第1个附件，在计划、实施和记录HPLC仪器的确认过程时，应将本文件与核心文件一起使用。

The core docume nt contains the gen eral in troduct ion and the Level I and II of qualificati on, com mon to all type of in strume nts, and the prese nt annex contains HPLC in strume nt-related recomme ndati ons on parameters to be checked and the corresp onding typical accepta nee Limits, as well as practical examples on the Methodology that can be used to carry out these checks.
核心文件包括了简介和第一阶段和第二阶段的确认，它们适用于所有类型的仪器。

本附件包括
了HPLC仪器相关的需要检查的参数和相应典型的可接受标准的推荐，以及可用于进行这此检查的实用方法学举例。

Whe n qualify ing HPLC equipme nt, it should be no ted that it is acceptable to check at Level III and IV several of the mentioned parameters at the same time in a combined test procedure (e.g. “ overall ” system performance test giving information on peak area precision, retention time precision, gradient reproducibility, etc).
在进行HPLC设备确认时，应注意也可以对第三阶段和第四阶段几个提到的参数采用联合检测程序同时进行检查(例如“全面”系统性能检查，同时给出峰面积精密度、保留时间精密度、梯度重复性等的信息)
Level III. Periodic and motivated instrument checks
第三阶段定期和专门的仪器检查
Examples of requirements for HPLC instruments and detectors
HPLC仪器和检测器要求举例
Level IV. In-use instrument checks
第四阶段在用仪器的检查
Examples of requirements for HPLC instruments with UV or DAD detectors 配备UV或DAD检测器的HPLC仪器的
ANNEX I 附件1
Level III. Periodic and motivated instrument checks
第三阶段定期和专门的仪器检查
This Annex contains practical examples of tests and their associated tolera nee Limits for several parameters related to the performa nee of the differe nt modules of a HPLC.
本附件包含的是检测HPLC仪器的不同模块性能的一些参数的实际例子及其参数的误差限度。

These examples can be con sidered by the OMCLs as possible approaches to perform the Level III of the equipment qualification process: “ Periodic and motivated instrument checks
OMCLs认为这些例子可以用来执行设备确认过程的阶段III “定期和专门的仪器检测”。

HPLC SOLVENT DELIVERY SYSTEM
HPLC溶剂传输系统
The followi ng tests are proposed for the periodic and motivated check of the HPLC solve nt delivery system: flow
rate and gradie nt test.
以下测试建议用于对HPLC溶剂传输系统的周期性主动检查：流速和梯度检测
FLOW RATE 流速
Materials:材料
Volumetric flask of 5 or 10 ml
5ml或10ml的容量瓶
Calibrated chrono meter
校正过的气压计
Sett in gs:设置
Mobile phase: degassed water
流动相：脱过气的水
No colu mn (ope n en d)*
无柱(开放式)*
Flow rate: adjusted betwee n 0.5 and 3.0 ml/m in
流速：调整到0.5至3.0ml/min之间
If high-pressure mixing systems are in stalled, this test has to be done on each solve nt cha nn el. 如果有安装高压混合系统，本测试应在每一溶剂通道进行。

* For certain equipment, e.g. in the case of low flow rates, the check would be performed by using a colu mn or a backpressure regulator.
*对于某些设备，例如，流速较低时，检测时需要用到一根色谱柱或背压调节器进行检查。

Method 方法:
Set the flow rate at an appropriate level and measure the time needed to fill the volumetric flask up to the mark. Record the time n eeded.
将流速设定在一个合适的水平，测量将容量瓶装满至刻度的时间。

记录所用的时间。

f = f = (VX 60)/t
....................... measured flow rate [ml/min] 测得的流速
t ..................... e lapsed time to fill up to mark [s]充满至刻度所需的时间
V .................... v olume of the volumetric flask [ml]容量瓶的体积
D= 100 X (f — F)/F
D .................... d eviation [%]偏差
F ..................... a djusted flow rate [ml/min] 调节后的流速
f ...................... m easured flow rate [ml/min] 测量的流速
Limits 限度：± 5%
GRADIENT COMPOSITION AND RIPPLE 梯度组成和波动
Setti ngs:设置
Stainl ess steel capillary e.g. 2000 x 0.12 mm in stalled in stead of a colu mn
不锈钢毛细管，例如2000X0.12mm,代替色谱柱安装于仪器
Detecti on: UV-Detector adjusted to 265 nm
检测器：紫外检测器调节至265nm
Mobile phase A: degassed water
流动相A :脱气水
Mobile phase B: degassed water containing 0.5% acet one
流动相B :含0.5%的脱气水
Flow rate: 1.0 ml/min
流速：1.0ml/分钟
Method 方法:
The test is carried out in the follow ing way by using a gradie nt program depe nding on the nu mber of solve nt cha nn els and the con figurati on of the system:
根据溶剂通道的数量和系统的配置，使用下列不同系统参数和梯度进行测试：
A-B
A-B and A-C
A-C, A-B and B-D
Start the test by pumping water for at least 10 min to equilibrate the system.
开始测试前，先用水平衡系统至少10分钟。

The zero % value at the start of the test is the baseli ne. All steps are measured at the beg inning of the horizontal part of the line either by software or manually on the paper print using a liner. The height of the 100% water/acet one mixture is used as the 100% value in the follow ing calculati on.
测试开始时的0%值是基线值。

所有步骤均在基线的水平部分开始时由软件或人工测量，并打印在纸上。

100%水/丙酮混合物的高度在以下计算中作为100%值。

%H = 100* h/H
%H ............... calculated compositi on 计算组成
h .................... height of the measured line 测量线的高度
H ................... height of the 100% water/acet one mixture line (mobile phase B)
100%水/丙酮混合物线的高度(流动相B)
d = %H —G
d .................... deviati on 偏差
G .................... gradie nt compositi on adjusted [% acet on e/water soluti on mixture = mobile phase B]
梯度组成调节【%丙酮/水溶液混合物二流动相B】
Limits 限度:
Absolute deviation: ± 2 of the adjusted value
绝对偏差：调整值的土2
The ripple of the gradie nt compositi on is the perce ntage of no ise of the 50% line from the gradie nt program. 梯度组分的波动为梯度程序噪声基线的50%
%R = 100* N /h 50
%R ................. ripple 波纹
h50 ....................... height of the 50% line 50% 线的高度
N ...................... h eight of the noise line, measured during 1 minute in the linear region 噪声线高度，在线
性区域1分钟期间测量的结果
Limits 限度:< 0.2 %
HPLC INJECTOR
HPLC进样器
Volume precisi on and carry-over are the tests proposed for the periodic and motivated check of the HPLC injector. 建议在HPLC进样器的定期和专门检查中进行容量(体积)精密度和残留检查。

VOLUME PRECISION AND CARRY-OVER
体积精密度和残留
Solutio ns:溶液
Solve nt A: metha nol: water R, 60 : 40.
溶液A :乙醇：水(试剂) 二60: 40
Refere nee soluti on (a): dissolve 15.0 mg methyl-, ethyl-, and propylparabe ne in 100.0 ml of solve nt A.
对照液(a):取15.0mg尼泊金M、尼泊金乙酯和尼泊金丙酯溶于100.0ml的溶剂A中
Refere nee soluti on (b): Dilute 1.0 ml of reference soluti on (a) to 10.0 ml of solve nt A.
对照液(b):取1.0ml对照液(a)用溶于10.0ml溶剂A中
Refere nee soluti on (c): Dilute 1.0 ml of reference soluti on (b) to 100.0 ml of solve nt A.
对照液(c):取1.0ml对照液(b)用溶于100.0ml溶剂A中
Sett in gs:设置
Column: Lichrospher 100 RP8, 5um, 125 x 4 mm, without precolu mn
色谱柱：Lichrospher 100 RP8, 5 um, 125 x 4 mm,无衬管
Mobile phase: metha nol : water = 60 : 40
流动相：乙醇：水二60: 40
Flow rate: 1.0 ml/min
流速：1.0ml/分钟
Detecti on: 254 nm
检测器：254nm
Injection volume: 20 口l
进样体积：20ul
Method 方法:
Injectio n scheme:进样序歹U
—6x referenee solution (b)对照液(b) 6针
—1x referenee solution (a) 对照液(a) 1 针
—1x solve nt A (bla nk injectio n 1) 溶剂A (空白1) 1 针
—1x referenee solution (b) 对照液(b) 1 针
—1x solve nt A (bla nk injectio n 2) 溶剂A (空白2) 1 针
—1x referenee solution (c) 对照液(c) 1 针
Limit 限度s:
Repeatability of peak areas: The relative standard deviation of the peak areas of all peaks in the chromatogram obtained with the referenee solution (b) should be < 1.0 %.
峰面积重复性：对照液(b)色谱图中所有峰的峰面积相对标准偏差应w 1.0 %
Carry-over: The perce ntage of the peak area corresp onding to propylparabe ne in the bla nk in jecti on 1 does not exceed 0.5% of 10 times the peak area of the propylparabe ne peak in the chromatogram obtained with the referenee solution (b) injected after the blank injection.
残留：尼泊金丙酯在空白图谱中的峰面积不超过在空白后进样的对照液( b)中尼泊金丙酯的对
应峰面积的10倍的0.5%
The perce ntage of the peak area corresp onding to propylparabe ne in the reference soluti on (c) is 0—1.1% of the
peak area of the propylparabe ne peak in the chromatogram obta ined with the reference 1st July 2011 Page 8 of 18
solution (b) injected after the blank injection.
在对照液(c)中尼泊金丙酯对应的峰面积为空白后进样的对照液( b)中尼泊金丙酯峰面积的
0.9-1.1%。

HPLC AUTOSAMPLER
HPLC自动进样器
Thermostatti ng accuracy and precisi on can be tested in the frame of the periodic and motivated check of the HPLC Autosampler.
对HPLC自动进样器的定期和专门检查可以检测温度的准确度和精确度。

THERMOSTATTING ACCURACY 温度准确度
Materials:材料
Calibrated temperature probe.
校正过的温度探头
Method 方法:
Select a temperature along the operational or required temperature range of the equipment. Wait until the system is equilibrated.
在可操作范围或要求的温度范围内选择并设定一个温度，等待系统平衡
By means of the calibrated probe, measure the actual temperature in the autosampler and compare it to the selected temperature.
采用校正过的温度探头，测量自动进样器的实际温度，与设定的温度进行比较
Repeat the same procedure at differe nt pre-selected points coveri ng the temperature ran ge.
在选定的温度范围内的不同温度点重复相同的步骤
Limit 限度s:
The actual temperature may not differ more than ± 3°C with respect to the selected temperature. 实际温度与设定的温度相比差值不超过土3°C
THERMOSTATTING PRECISION 温度精确度
Materials:材料
Calibrated temperature probe.
校正过的温度探头。

Method 方法:
Select a temperature along the operational or required temperature range of the equipment. Wait until the system is equilibrated.
选择一个操作温度或要求的仪器温度范围，等待系统平衡。

By means of the calibrated probe, make “ n” measu e mstiatelioV e daDeriod of time.
Compare the mean of the “ n” measurements to the selected temperature.
采用校正过的温度探头，在预设的时间内进行n次测量，比较n次测量值的平均值与设定的温度。

Limits 限度:
The actual temperature may not differ more than ± 3°C with respect to the selected temperature. 实际温度与设定的温度之间差值不超过土3°C
HPLC OVEN/COOLING DEVICE 色谱仪柱温箱/降温设施
Thermostatti ng accuracy is the parameter tested in this example of periodic and motivated check of the HPLC ove
n/cooli ng device.
温度的准确度是对HPLC柱温箱/降温设施进行定期和专门检查时检测的参数。

THERMOSTATTING ACCURACY 温度准确性
Materials:材料
Calibrated thermometer.
校正过的温度计
Method 方法:
Set the column oven temperature to 40 C, wait about 30 minutes to equilibrate the system, put a calibrated thermometer into the ove n and read the temperature after 10 minu tes.
设定柱温箱温度为40 等待约30分钟平衡系统，将校正过的温度计放入柱温箱中，在10分钟
后读取温度。

Limits 限度:38 - 42 C.°
HPLC UV/DAD DETECTOR
HPLC紫外/DAD检测器
The periodic and motivated check of the HPLC UV/DAD detector can be performed by test ing the lin earity and the wavele ngth accuracy.
对HPLC紫外/DAD检测器的定期和专门的检查，可以对线性和波长准确性进行检测。

LINEARITY 线性
Solutions:溶剂配制
Std. 1: 0.5 ⑷ caffeine/1 ml metha nol HPLC Grade
标准1: 0.5⑷咖啡因/1ml色谱级甲醇
Std. 2: 1.0 (jg caffein e/1 ml metha nol HPLC Grade
标准2: 1.0⑷咖啡因/1ml色谱级甲醇
Std. 3: 5.0 jg caffeine/1 ml metha nol HPLC Grade
标准3: 5.0 jg咖啡因/1ml色谱级甲醇
Std. 4: 25.0 jg caffei ne/1 ml metha nol HPLC Grade
标准4: 25.0 jg咖啡因/1ml色谱级甲醇
Std. 5: 50.0 jg caffei ne/1 ml metha nol HPLC Grade
标准5: 50.0⑷咖啡因/1ml色谱级甲醇
Std. 6: metha nol HPLC Grade (bla nk)
标准6:色谱级甲醇(空白)
Std. 5: weigh 9.0 to 11.0 mg caffeine and fill up to 200.0 ml with methanol HPLC
标准5:称取9.0至11.0mg咖啡因，用HPLC级别甲醇稀释至200.0ml
Std. 4: dilute 50.0 ml of Std. 5 to 100.0 ml with metha nol
标准4:取50.0ml标准5溶液用甲醇稀释至100.0ml
Std. 3: dilute 10.0 ml of Std. 5 to 100.0 ml with metha nol
标准3:取10.0ml标准溶液5用甲醇稀释至100.0ml
Std. 2: dilute 20.0 ml of Std. 3 to 100.0 ml with metha nol
标准2:取20.0ml标准3溶液用甲醇稀释至100.0ml
Std. 1: dilute 10.0 ml of Std. 3 to 100.0 ml with metha nol
标准1:取10.0ml标准3用甲醇稀释至100.0ml
Sett in gs:设置
Colum n: RP-18 5 um 30-50 x 2,1-4.6 mm or capillary 2000 mm x 0.12 mm ID
色谱柱：RP-185 um 30-50 x 2,1-4.6 mm or 毛细管柱2000 mm x 0.12 mm ID
Mobile phase: metha nol HPLC Grade
流动相：色谱级甲醇
Oven temperature: 40 C °
柱温箱温度：40 C
Flow rate: 1.0 ml/m in (adjusted by using 100% metha nol)
流速：1.0ml/min (用100%甲醇调整)
Detectio n: 273 nm
检测波长：273nm
Injection volume: 20 口l
进样量：20ul
Method 方法:
Injection scheme:
进样序列
2 x bla nk 2针空白
1 x Std. 1
1 x Std. 2
1 x Std. 3
1 x Std. 4
1 x Std. 5
标准1-5依次各一针
Limits 限度:r2> 0.999
Remark: As this test employs different test solutions to be injected, it covers also the check of correct positioning vials in the autosampler.
注：由于此检测中要进样不同供试溶液，因此也包括了对自动进样器样品瓶位置正确性的检查。

WAVELENGTH ACCURACY 波长准确度
If there are built-i n test procedures for the determ in ati on and adjustme nt of wavele ngth accuracy, follow the in struct ions of the in strume nt manu al.
如果内置检测程序可以检测和调节波长准确度，则根据操作手册进行操作
In all other cases use the procedure described below.
否则，就采用以下程序
Solutio ns:溶液
DAD: caffeine Std. 5 from the linearity testing 线性测试中的咖啡因5号标准溶液
UV/VIS: 1.0 J g/ml anthracene solution in water 1.0」g/ml 的蔥水溶液
Sett in gs:设置
Mobile phase: 15% acet on itrile in water
流动相：15%乙腈水溶液
Column： RPI8： 5 rn 30-50 x 2,1-4.6 mm or capillary 2.0 m x 0.12 mm ID
色谱柱：RP18, 5 口m, 3050 >2.1-4.6mm或毛细管柱20rriX0.12mm内径
Oven temperature: 40 C °
柱箱温度：40 C
Flow rate: 1.0 ml/min (adjusted by using 15% acet on itrile in water)
流速：1.0ml/min (用15%的乙腈水溶液调节)
Detection: scan from 230 nm to 290 nm (DAD)
检测：自230至290nm扫描(DAD )
Injection volume: 20 (jl
进样体积：20 口l
Method 方法:
DAD: Inject 20 M of the caffeine solution and record the spectrum. The maximum is at 272 nm and the mi nimum at 244 nm.
DAD:咖啡因溶液进样20M，记录色谱图，最大吸收波长为272nm,最小为244nm.
UV/VIS: fill the cell with the anthracene solution and change the wavelength from 248 to 254 nm in 1 nm steps; record the maximum of absorption. The theoretical value is 251 nm.
UV/可见光：向池中注入蔥溶液，将波长以1nm的步距从248改为254nm,记录最大吸收值。

理论值为251 nm。

Limits 限度:< 2 nm
HPLC FLUORESCENCE DETECTOR
HPLC荧光检测器
The follow ing three parameters are proposed for the performa nee of the periodic and motivated check of the HPLC fluoresce nt detector:
下面三个参数建议作为对荧光检测器性能进行定期和专门检查时的参数。

WAVELENGTH ACCURACY EXCITATION 波长激发准确度
Method 方法:
Rinse and fill the measuring cell with de-ionized water
用去离子水淋洗并充满检测池
Adjust the excitati on wavele ngth to 350 nm.
调节激发波长为350nm
Measure the emissi on and subtract 397 nm (theoretical value).
测量发射波长并减去397nm (理论值)
Limits 限度:± 3 nm
WAVELENGTH ACCURACY EMISSION 波长发射准确度
Method方法:方法
Rinse and fill the measuring cell with de-ionized water
用去离子水淋洗并充满检测池
Adjust the emissi on wavele ngth to 397 nm.
调节发射波长为397nm
Measure the excitati on and subtract 350 nm (theoretical value).
测量激发波长并减去350nm (理论值)
Limits 限度:± 3nm
SENSITIVITY 灵敏度
Solutio ns:溶液
Quinine HCl.2H 2O solution cone. 0.015 口g/ml (=15 ppb)
二水盐酸喹啉溶液，浓度0.015卩g/ml (=15 ppb)
The quinine soluti on is prepared with the follow ing mobile phase: dissolve 6.8 g of potassium dihydroge n phosphate R and 3.0 g of hexylamine R in 700 ml of water R, adjust to pH 2.8 with dilute phosphoric acid R, add 90 ml of aceto nitrile R and dilute to 1000.0 ml with water R.
喹啉溶液用以下流动相制备：溶解 6.8g磷酸二氢钾和3.0g己胺于700ml试剂级水中，用稀磷酸调pH至2.8,
加90ml乙腈并用试剂级水稀释至1000.0ml.
Sett in gs:设置
The chromatographic con diti ons are set accord ing to Ph. Eur. “ Quinine HCl ”
(01/2005:C “ Other cinchona alkaloids ” , with modified flow rate an(clea c oticetrtration. 色谱条件设定依
据欧洲药典“盐酸喹啉” (01/2005:0018),检测“其他金鸡纳生物碱”，用不同的流速和乙腈浓度。

Mobile phase: as above
流动相：同上
Column: RP18, 5 pm, 250 x 4.6 mm
色谱柱:RP18, 5 p m, 25X 4.6 mm
Flow rate: 1.2 ml/min
流速：1.2 ml/min
Excitati on wavele ngth: 350 nm
激发波长：350nm
Emissi on wavele ngth: 397 nm
发射波长：397nm
Flow-cell volume: 8 M (for this example, a Waters 2475 Multi Fluorescentie detector was used.
Flow-cell volume may vary depe nding on the in strume nt manu facturer)
流动池体积：8M1此例中，使用的是Waters2475多荧光检测器，流动池体积可能因不同的仪器厂商而不同) Method 方法:
Inject 10 M bf the quinine solution and measure the peak height.
进样喹啉溶液10口」测量峰高度
Inject 10 M of the blank and measure the peak height of the noise.
进样空白溶液10口」测量噪音峰高
Divide the peak height of the quinine solution by 3 times the peak height of the noise.
将喹啉溶液峰高除以3倍噪音峰高
Divide the concentration of the quinine solution by the previously obtained factor.
将喹啉溶液浓度除以之前的系数
Limit 限度:< 0.5 ppb
HPLC ELECTROCHEMICAL DETECTOR
HPLC电化学检测器
Accuracy and stability of the signal are the proposed parameters to be tested during the periodic and motivated check of the HPLC electrochemical detector.
电化学检测器的信号准确度和稳定性是对其定期和专门检测中建议检测的参数。

ACCURACY AND STABILITY OF THE SIGNAL 信号准确度和稳定性
Setti ngs:设定
Cell pote ntial of a dummy cell: 800 mV
空白池电压：800mv
Rise time filter: 0.1 s
增加时间过滤：0.1s
Ran ge: 0.1 nA
范围：0.1nA
Temperature: 30 C °
温度：30C
Method 方法:
Accuracy: Measure the electric curre nt and subtract 2.67 nA (theoretical value)
准确度：检测电流并减去2.67nA (理论值)
Stability: Measure the noise over a period of 5 minutes
稳定性：连续5分钟测量噪音
Limits 限度:
Accuracy (cell current): 0.05 nA
准确度(池电流)：0.05nA
Stability of the sig nal (Noise): max. 2 pA or 20 mV
信号(噪音)稳定性：最大2pA或20mV
HPLC RID DETECTOR
HPLC的RID (折射率)检测器
Signal to Noise ratio and drift over time are the parameters proposed for the periodic and motivated check of the HPLC RID (refractive in dex) detector.
HPLC的RID (折射率)检测器的信噪比和时间漂移是定期和专门检测中建议检测的参数
SIGNAL TO NOISE RATIO 信噪比
Solutio ns 溶液:
Stan dard soluti on: D-fructose concen trate soluti on at 4.0 mg/ml (dilute 200.0 mg fructose + 20 ml water + 25.0 ml acetonitrile up to 50.0 ml with water for HPLC)
标准溶液：D-左旋糖溶液4.0mg/ml (将200.0mg左旋糖+20ml水+25.0ml乙腈，用色谱纯水稀释至50.0ml)
Setti ngs设置:
Colu mn: spherisorb NH2 (or equivale nt) 250 x 4.6 mm or other
色谱柱：spherisorb NH2 (或相当的)250 x4.6 mm 或其他
Oven temperature: 38 C °
柱温箱温度：38 C
Flow rate: 1.0 ml/min
流速:1.0 ml/min
Injection volume: 20 (jl
进样量:20 口l
Mobile phase: 0.253g sodium hydroge n phosphate R in 220 ml + 780 ml acet on itrile 流动相：0.253g磷酸氢二钠溶于220ml+780ml乙腈
Method 方法:
After equilibration, inject three times a blank solution of mobile phase over a run time where the system is stable. Measure the baseline noise over an appropriate period.
平衡后，以流动相为空白进样三针，系统稳定运行适当时间，测定适当时长内噪音基线
The baseli ne no ise is accepted if the mean height of the three replicates is < 100QV.
可接受基线噪音：三次平行进样平均值< 1000 ^V
To calculate the signal to noise ratio, inject three times a solution of fructose at 0.4 mg/ml and calculate the mean of the three replicates.
计算信噪比，左旋糖溶液0.4mg/ml进样三针，计算平均值
Limit 限度:S/N
限度：信噪比＞10
DRIFT OVER A DEFINED PERIOD OF TIME 指定时长的漂移
Method 方法:
Calculate the slope of the amplitude of random variations in the detector ' s signal over 计算检测器信号在1分钟内随机波动幅度的斜率
Limits 限度:±0.1 mV/min
Alter natively, the requireme nt may be expressed in △ RI/min or in % of full scale of the selected
ran ge.
或者，也可以表述RI/min或者所选量程的全范围的%。

HPLC CD DETECTOR
HPLC的CD检测器
The follow ing tests are proposed to perform the periodic and motivated check of the HPLC CD
(circular dichroism) detector.
对HPLC的CD (圆二图谱)检测器进行定期和专门检查时，建议进行以下检测。

LINEARITY AND SIGNAL TO NOISE RATIO
线性和信噪比
Solutio ns:溶液
Refere nee soluti on (a): dissolve 25.0 mg D(-) pan tolact one in 50.0 ml water
对照溶液(a):溶解25.0mg D(-)泛酰内酯于50.0ml水中
Refere nee soluti on (b): dilute 2.0 ml of refere nee soluti on (a) to 10.0 ml with water
对照溶液(b):取对照溶液(a)2.0ml用水稀释至10.0ml
Refere nee soluti on (c): dilute 4.0 ml of refere nee soluti on (a) to 10.0 ml with water
对照溶液(c):取对照溶液(a)4.0ml用水稀释至10.0ml.
Refere nee soluti on (d): dilute 6.0 ml of refere nee soluti on (a) to 10.0 ml with water
对照溶液(d)：取对照溶液(a)6.0ml用水稀释至10.0ml.
Refere nee soluti on (e): dilute 8.0 ml of refere nee soluti on (a) to 10.0 ml with water
对照溶液(e):取对照溶液(a)8.0ml用水稀释至10.0ml.
Refere nee soluti on (f): dilute 0.5 ml of refere nee soluti on (b) to 25.0 ml with water
对照溶液(f)：取对照溶液(b)0.5ml用水稀释至25.0ml.
Setti ngs:设定
Column 色谱柱:C18, 150 4 mm, 5 (jm
Mobile phase 流动相:aceto nitrile 乙腈:water水= 10 : 90
Flow 流速:1.0 ml/min
Detection 检测波长:225 nm
Injection volume 进样量:20 j
Method 方法:
Check the linearity of de CD- and UV-signal of D(-)pantolactone referenee solution a,b,c,d,e.
检查CD和UV信号与D(-)泛酰内酯对照溶液a,b,c,d,e的线性关系
Measure the no ise of the CD-sig nal of refere nee soluti on ⑴ betwee n 0—10 min.
检查0-10分钟内对照溶液⑴的CD信号的噪声
-Calculate the absolute concen trati on (4) in the cell
计算池中绝对浓度(口g)
-Calculate the signal-to-noise ratio (S/N) for 0.01 旧in the cell
计算池中0.01 信噪比(S/N)
-Calculate the sen sitivity with the calculated S/N and the specified S/N二2 (0.01 x 2/ S/N calculated) -计算所得信噪比及信噪比为2(0.01 X 2/ S/N calculated)时的灵敏度
Limits 限度:
Linearity: The linearity of the calibration line obtained with reference solution (a,b,c,d,e) should be r > 0.999. 线性：由对照液(a,b,c,d,e)所得的校正曲线的线性应是r> 0.999。

Sen sitivity: The sen sitivity at S/N= 2 should be better than 0.01 4.
灵敏度：信噪比为2时灵敏度应好于0.01 口g
S/N ratio: The Limit for S/N is > 1.0 and the sensitivity should be 0.020 旧at maximum.
信噪比：信噪比的限度为〉1.0时，灵敏度最大值为0.020 口g
DRIFT OVER A DEFINED PERIOD OF TIME 指定时长的飘移
Setti ngs设定:
Column 色谱柱:C18, 150 x 4 mm, 5 pm
Mobile phase 流动相:aceto nitrile 乙腈:water水= 10 : 90
Flow 流速:1.0 ml/min
Detection 检测波长:290 nm
Injection volume 进样量:20 口l
Method 方法:
Inject water and stop the flow after 5 minutes. Measure the CD-signal for 1 hour.
进水样，5分钟后停止流动。

测量CD信号，持续1小时。

Measure with the cursor the drift of the baseline between 5 and 65 min.
用指针测量5到65分钟之间的基线飘移
Limits 限度:
Not more than 0.1 mdeg/h
不超过0.1 mdeg/h
SPECTRA COMPARISON 谱图比较
Solutio ns 溶液：
Refere nee soluti on (a): dissolve 5.0 mg dexamethas one in 10.0 ml 40% acet on itrile 对照溶液(a):溶解5.0mg地塞米松于10.0ml 40%乙腈中。

Setti ngs设定:
Column 色谱柱:C18, 150 x 4 mm, 5 口m
Mobile phase 流动相:aceto nitrile 乙腈:water水= 40 : 60
Flow 流速:1.0 ml/min
Detection 检测波长:230 nm
Injection volume 进样量:20ul
Method 方法:
比较检测器安装结果(见下表)
Limits 限度:
The maxima and minima may not differ more than 4 nm. 土最大和最小值之差不可超过4nm。