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دستگاه اسپکتروفتومتر یا طیف سنج نوری ابزاری اپتیکی است که با عبور دادن نور از مواد در طول موج های مشخصی، به شناسایی برخی از ویژگی های مولکولی آنها می پردازد. اسپکتروفتومتر، یکی از ابزارهای کلیدی و قلب هر آزمایشگاه تحلیلی است.
در حقیقت، اسپکتروفتومتر ها، تعداد یا شدت فوتون های جذب شده توسط ماده را پس از عبور از محلول نمونه اندازه گیری می کند. با استفاده از این دستگاه، نوع ترکیبات یک ماده شیمیایی شناخته شده و مقدار آنها (غلظت) را می توان تعیین کرد.
امروزه دستگاه های اسپکتروفتومتر، ابزارهای تحلیلی قدرتمندی هستند که در حوزه های مختلف علمی و صنعتی از جمله شیمی، زیست شناسی، پزشکی و علوم محیطی استفاده می شوند. این ابزارهای کلیدی، با اندازه گیری جذب، گسیل یا پراکندگی نور در طول موج های خاص امکان تجزیه و تحلیل دقیق و سریع نمونه ها را فراهم می کنند.
در هنگام خرید اسپکتروفتومتر، نیاز است که اطلاعات جامعی از عملکرد این دستگاه و مشخصات فنی ان داشته باشید. برای اطلاعات بیشتر و آشنایی با عملکرد و قابلیت های این دستگاه کاربردی با ما همراه باشید.
اسپکتروفتومتر، به شکل مدرن امروزی، در سال 1940 توسط آرنولد بکمن و همکارانش ساخته شد و با گذشت زمان، به اشکال پیچیده تری تکامل یافت و معرفی ابزارهای پراکننده نور و مبتنی بر منشور، تجزیه و تحلیل طیفی دقیق تری را برای اسپکتروفتومتر فراهم کرد.
در نهایت، دستگاه های اسپکتروفتومتر مبتنی بر توری پراش رایج شدند و دقت و تطبیق پذیری بهتری را ارائه کردند و به عنوان دستگاهی استاندارد برای آنالیز مواد، در بسیاری از آزمایشگاه های شیمی شناخته شدند.
تعاریف متعددی برای دستگاه های اسپکتروفتومتر وجود دارد، اما به زبان ساده، اسپکتروفتومتر ابزاری است که به دانشمندان و محققان کمک می کند تا خواص نور و نحوه تعامل آن با مواد مختلف را مطالعه کنند.
به عبارت دیگر، بسته به اینکه نور چه تعاملی با ماده دارد، نور فرودی اولیه با نور پس از برهمکنش دیگر یکسان نیست و دچار تغییراتی می شود. با بررسی و آنالیز این نور تغییر یافته، می توان فهمید که ماده ای که با آن برهمکنش داشته است، چه ویژگی هایی دارد.
به دلیل ابعاد ریز و کوانتومی اتم ها و مولکول ها، نمی توان آنها را با ابزارهای معمولی مشاهده کرد. بنابراین، اسپکتروفتومتر دستگاهی است که این امکان را به شما می دهد که بتوانید بدون مشاهده مستقیم داخل اتم یا مولکول، ویژگی آنها را تعیین کنید.
به دلیل همین مشخصه های منحصر به فرد اسپکتروفتومتر است که به طور گسترده ای برای آنالیزهای کمی (عددی) و کیفی، مانند تعیین غلظت یک ماده خاص در یک محلول استفاده می شود.
کاربرد اسپکتروفتومتر تنها به موارد کمی محدود نمی شود، بلکه فراتر از این ها، می تواند برای تجزیه و تحلیل های کیفی، از جمله شناسایی مواد بر اساس طیف جذب آنها نیز به کار رود. این ویژگی، بیشتر در بخش های کنترل کیفی محصولات مختلف در صنایع مفید است.
علاوه بر این، اسپکتروفتومتر ها نقش مهمی در کاربردهای متعددی از جمله تجزیه و تحلیل DNA، کشف دارو، نظارت بر محیط زیست، کنترل کیفیت آب و صنایعی مانند تولید مواد غذایی دارند.
دستگاه های اسپکتروفتومتر، ابزاری مفید برای آموزش خواص نور و ماده نیز هستند. برای مثال، با استفاده از یک دستگاه اسپکتروفتومتر، افراد می توانند طیف جذب یک محلول را ببینند و با آنالیز آن با ترکیب و خواص مواد آشنا شوند.
در بخش قبل گفته شد که اساس عملکردی یک سیستم اسپکتروفتومتر، برهمکنش نور و ماده است. زمانی که تابش الکترومغناطیسی به ماده ای می تابد، بسته به حالت ماده، ضخامت و ترکیب شیمیایی ماده، می تواند به روش های مختلفی با ماده تعامل کند، از جمله جذب و عبور، بازتاب و پراکندگی.
وقتی که یک ماده در معرض تابش نور الکترومغناطیسی قرار می گیرد، اگر بسیار شفاف باشد، می تواند کل نور یا بخشی از آن را از خود عبور دهد. در مقابل اگر ماده ای تیره باشد، می تواند نور را جذب کند و اگر ماده براقی مانند فلز باشد، آن را بازتاب می کند.
برهمکنش از نوع پراکندگی نیز زمانی رخ می دهد که ذرات کوچک یا بی نظمیهای موجود در سطح یک ماده باعث تغییر جهت نور شود. برای جزئیات بیشتر در مورد هر کدام از این برهمکنش ها، بهتر است به مقاله “فرایند های جذب و عبور” مراجعه نمایید.
اسپکتروفتومتر ها نیز بر اساس نوع تعامل نور و ماده، متفاوت عمل می کنند و با اندازه گیری مقدار نور جذب شده یا بازتاب آن توسط یک نمونه (ماده مورد آنالیز) در طول موج های مختلف، اطلاعات ارزشمندی در مورد ترکیب و خواص آن ارائه می دهند.
بنابراین بر اساس نحوه برهمکنش نور و ماده، دو گروه کلی از اسپکتروفتومتر وجود دارد و بر همین اساس، اجزای تشکیل دهنده آنها نیز می تواند متفاوت باشد.
انواع برهمکنش نور با ماده
در مورد یک دستگاه اسپکتروفتومتر نوع جذبی-عبوری، ایده اصلی این است که نور از یک ماده (معمولاً یک محلول یا یک ماده شفاف) عبور داده می شود و میزان جذب و عبور نور توسط ماده مورد مقایسه و ارزیابی قرار می گیرد.
در این نوع از اسپکتروفتومتر، بخشی از نور توسط مولکول های تشکیل دهنده ماده مورد نظر جذب شده و مابقی آن، از نمونه عبور می کند. شدت پرتو قبل و پس از عبور از آن ماده اندازه گیری و ثبت می شود. با مقایسه این شدت ها، ویژگی های کلی یک نمونه، مانند غلظت و یا نوع ترکیبات آن مشخص می شود.
جذب و عبور دو مفهوم کلیدی هستند که باید به طور دقیق تعریف شوند. اسپکتروفتومتری نوع جذبی، تکنیکی است که برای اندازه گیری میزان قابلیت ماده در عبور دادن نور به کار می رود، در حالی که عبور، میزان نور انتقال یافته توسط ماده نمونه را اندازه گیری می کند.
در طیف سنجی، روش جذبی-عبوری مکمل یکدیگر هستند و بر این مبنا عمل می کنند که هر مولکول یا ماده، می تواند طیف خاصی از امواج الکترومغناطیسی را جذب کند و مابقی طیف بدون اینکه تغییری در ماده ایجاد کند، از آن عبور می کند. بنابراین، هر دو پدیده را همزمان می توان در یک دستگاه به کار برد.
فرایند جذب زمانی اتفاق می افتد که نور به شکل دیگری از انرژی تبدیل می شود و هنگام عبور از یک محیط ناپدید می شود. در حالی که در فرایند عبور، نور می تواند به راحتی از یک محیط دیگر عبور کند.
بسته به نوع مولکول، تعداد الکترون ها و به تبع آن، آرایش الکترونی و پیوند های شیمیایی داخلی هر ماده متفاوت است و از این رو، طول موج های خاصی را بیشتر از بقیه جذب می کنند.
دستگاه اسپکتروفتومتر که امواج الکترومغناطیسی را آنالیز و آشکارسازی می کند، باید طوری طراحی شود که در فرکانس یا طول موج جذب ماده مورد آزمایش، فعال باشد. به عبارتی، یک دستگاه اسپکتروفتومتر باید بتواند براساس نوع برهمکنش نور با ماده (جذب و عبور)، در طول موج های فعال مواد مورد بررسی، آنالیز های لازم را انجام دهد.
عملکرد نهایی دستگاه اسپکتروفتومتر به گونه ای است که در خروجی، توابعی از شدت نور برحسب طول موج یا فرکانس گزارش داده می شود (طیف جذب یا عبور ماده مورد آنالیز).
طیف های جذب و عبور
این طیف ها اطلاعات مهمی را در مورد ساختار مولکولی موادی که امواج الکترومغناطیسی روی آنها متمرکز شده است، در اختیار ما قرار می دهند. به عبارتی، «اثر انگشت» یا مشخصه عناصر و ترکیبات مختلف هستند.
اسپکتروفتومتر ایرانی نوع جذبی-عبوری OPAL
در یک اسپکتروفتومتر، برای محاسبه میزان جذب نور توسط نمونه، از قانون بییر-لامبرت استفاده می شود. میزان عبور نور از درون یک محلول یا یک ماده شفاف، به مقدار نوری اطلاق می شود که به طور کامل از نمونه عبور کرده و به آشکارساز برخورد می کند.
(1) T=I/Io
I0، شدت نور تابشی به ماده نمونه و I، شدت نور عبوری از نمونه است. دقت داشته باشید که مقادیر عبور عمدتاً به صورت درصد محاسبه می شوند.
در این نوع دستگاه ها، معمولاً برای اندازه گیری و محاسبه میزان جذب و عبور نمونه ها، از قانون بیر-لامبرت استفاده می شود و می توان از آن برای تعیین غلظت نمونه (c) استفاده کرد.
در اندازه گیری جذب، نور از درون یک کووت حاوی ماده نمونه مورد تحقیق عبور می کند و شدت نور پس از عبور از کووت با نور پیش از عبور مقایسه می شود.
هر چه کووت پهن تر باشد، نور توسط مولکول های بیشتری از نمونه بیشتری جذب شده و در مقابل، نور عبوری کمتری خواهیم داشت. بنابراین، میزان جذب نور در ماده نمونه، به طول مسیر عبوری نور (L) وابسته است. بر طبق قانون بیر-لامبرت داریم:
(2) A= ϵLc
A، میزان جذب نور توسط ماده است و ϵ ضریب تضعیف مولی یا ضریب خاموشی شناخته می شود که بسته به ماهیت هر ماده، متفاوت است.
نور عبوری از نمونه
میزان جذب، با لگاریتم نسبت شدت نور پرتوهایی است که وارد نمونه شده و از نمونه خارج می شوند، متناسب است.
(3) A = log10 (I0/I)
میزان جذب مختلف توسط مواد نمونه
بنابراین، طبق معادله (1)، مقدار جذب را می توان بر اساس میزان عبور بیان کرد:
(4) A=-log10 (T)
طبق معادله بالا مشخص است که وقتی تمام نوری که به جسم می رسد، جذب آن شود، مقدار عبور 0٪ و مقدار جذب 100٪ است و بالعکس.
مقادیر جذب و عبور در اسپکتروفتومتر
توجه داشته باشید که در بسیاری از دستگاه های اسپکتروفتومتر مدرن امروزی، هر دو قابلیت جذب و عبور به طور همزمان مورد آنالیز قرار می گیرد و هر زمان که بخواهید می توانید دستگاه را بر روی حالت جذب یا عبور تنظیم کنید.
بر اساس معادلات بخش قبل، نمودار معمولی که قانون بیر-لامبرت را نشان می دهد همواره خطی و همبستگی مثبت خواهد داشت که محور x واحد غلظت و محور y مقدار جذب را نشان می دهند.
در معادله خطی (2) معمولاً دو متغیر دیگر، یعنی ضریب خاموشی مولی و طول مسیر نوری، هر دو ثابت فرض می شوند.
طول مسیر نوری که همواره ثابت است و در مورد ضریب مولی، از آنجایی که شرایط محیطی و دمایی تغییر نمی کند و ماده نمونه نیز تغییر ماهیتی خاصی ندارد، فرض می شود که ثابت است.
با افزایش غلظت ماده، قدرت جذب آن نیز افزایش می یابد. این الگو منطقی است زیرا اگر غلظت افزایش یابد، به این معنی است که مولکول های بیشتری برای جذب نور وجود دارند و باعث افزایش میزان جذب نور می شود.
نمودار خطی غلظت در اسکتروفتومتری جذبی
توسعه اسپکتروفتومتر های نوع جذبی، تحقیقات علمی را متحول کرده است و امکان تجزیه و تحلیل کمی و کیفی طیف گسترده ای از مواد و پدیده ها را فراهم کرده است. تکامل مداوم قطعات اپتیکی و الکترونیکی، آنها را به ابزاری ضروری در آزمایشگاههای مدرن تبدیل کرده است و کاربرد های گوناگونی دارند.
برای تعیین غلظت ترکیبات نامشخصی از یک محلول، می توان از طیف های جذبی استفاده کرد و داده های جذب مجموعه ای از محلول های استاندارد با غلظت های مشخص، یک منحنی جذب استاندارد را ایجاد می کند.
با اندازه گیری و مقایسه منحنی جذب ماده مجهول با این منحنی های استاندارد، غلظت آن تعیین می شود که بر مبنای قانون بیر-لامبرت عمل می کنند.
آنالیز آب توسط اسپکتروفتومتر تک پرتویی AURORA
هر ماده خالص بسته به ساختار الکترونی و مولکولی آن، طیف جذب منحصر به فردی خواهد داشت. بنابراین، یک طیف جذبی به شناسایی مواد ناشناخته کمک می کند.
برخی از گروه های عاملی (یا به عبارتی نوع پیوندهای شیمیایی) دارای طیف های جذبی متفاوتی هستند.
شناسایی آنها در یک طیف، نشان دهنده این است که آیا آن گروه عاملی در ترکیب وجود دارد یا خیر. بعلاوه، نشان دهنده موفقیت آمیز بودن یک واکنش در یک آزمایش به خصوص نیز می باشد.
اسپکتروفتومتر بازتابی، ابزاری اپتیکی است که برای اندازه گیری خواص بازتابی مواد (خواص سطحی یک ماده) در طیف وسیعی از طول موج ها استفاده می شود و میزان نور بازتاب شده از سطح نمونه را ارزیابی می کند.
فرایند بازتاب نور، بسته به نوع سطح و طول موج نور تابیده شده به سطح، در فرایندهای مختلفی می تواند انجام شود و هر فرایند، کاربرد ویژه خود را دارد و اگر سطوحی را آنالیز می کنید که کاملاً مات هستند، یک اسپکتروفتومتر بازتابی می تواند این کار را به خوبی انجام دهد.
از آنجایی که ابزار های بازتابی طیفی نور منعکس شده را می گیرند، دقیق ترین و قابل تکرارترین نتایج را برای سطوح صاف و مات ارائه می کنند.
برای مثال، در یک نقاشی، الیاف یا یک بافت پوستی، رنگ ها سطوح زیرلایه را می پوشانند و بنابراین نمی توان به طور دقیق بخش های زیرین را مورد قیاس چشمی قرار داد و این رنگ ها، کاندیدای خوبی برای اندازه گیری بازتاب هستند.
طیف حاصل از یک اسپکتروفتومتر نوع بازتابی
شماتیک کلی از عملکرد یک اسپکترفتومتر نوع بازتابی
انواع مختلف اسپکتروفتومتر، بر اساس کاربرد و عملکرد مورد نظر، همگی با یکدیگر متفاوت هستند اما در کلیت، اجزا و عملکردهای مشابهی دارند که در ادامه به آنها خواهیم پرداخت. به طور کلی در یک اسپکتروفتومتر:
شماتیک کلی از عملکرد یک اسپکتروفتومتر جذبی-عبوری
اسپکتروفتومتر ها شدت نور را در طول موج های مختلف اندازه گیری می کنند و برای اطمینان از اندازه گیری های دقیق، استفاده از منابع نوری مناسب ضروری است و در ادامه برخی از منابع نوری رایج برای اسپکتروفتومتر ها آورده شده است:
این لامپ ها طیف پیوسته ای از نور مرئی و فروسرخ نزدیک را تأمین می دهند و این گروه از منابع نوری، خروجی پایداری را در طول زمان ارائه می دهند و برای اسپکتروفتومتر چند منظوره مناسب هستند.
لامپ های دوتریوم نور فرابنفش (UV) را در محدوده 190-400 نانومتر ساطع می کنند و معمولاً به عنوان منبع بخش UV در ترکیب با یک لامپ تنگستن-هالوژن، برای پوشش طیف طول موجی کامل استفاده می شوند.
لامپ های زنون طیف وسیعی از نور از جمله نواحی UV، مرئی و مادون قرمز را تولید می کنند و آنها خروجی با شدت بالایی دارند و اغلب برای برنامه هایی که نیاز به حساسیت بالا یا اسکن سریع است، استفاده می شوند.
با این حال، میزان نور سرگردان ابزاری در آنها بالاتر است و انرژی کمتری در انتهای مرئی دور وجود دارد. با این وجود، برای اندازه گیری های عمومی ایده آل هستند.
LED ها در طول موج های مختلفی در دسترس هستند که آنها را برای کاربرد های ویژه (بازه های طول موجی کم) مناسب می کند. آنها مزایایی مانند مصرف انرژی کم، طول عمر طولانی و اندازه کوچک را ارائه می دهند.
این لامپ ها خطوط قوی در طول موج های خاصی در نواحی UV و مرئی ساطع می کنند و معمولاً به دلیل خطوط انتشار قوی، به عنوان منابع کالیبراسیون شناخته می شوند و کاربرد دارند.
در اسپکتروفتومتر های قدیمی، از منشورها به عنوان تجزیه کننده نور استفاده می شد، اما امروزه تقریباً در تمامی انواع مدرن از توری پراش برای تولید طیف طول موجی (تفکیک نور) استفاده می کنند و بر روی پیکسل های مختلف آشکارساز پخش می شود.
از جمله مزایای دستگاه اسپکتروفتومتر مجهز به توری پراش (در مقایسه با منشورها) می توان به موارد زیر اشاره کرد:
توری های پراش، طبق تعریف از خطوط یا شیار بر روی یک بستر تشکیل شده اند که چگالی این خطوط است که معیار میزان تفکیک کنندگی توری است و باید متناسب با سیستم آشکارساز انتخاب شوند.
اگر چگالی خط توری بیش از حد مجاز سیستم آشکارسازی باشد، نور در نواحی اطراف آشکارساز نیز پخش می شود و در نتیجه دقت آنالیز را کم می کند و در صورت کم بودن چگالی نیز تمامی طیف پوشش داده نمی شود.
تأثیر چگالی خط توری پراش در آنالیز نهایی یک اسپکتروفتومتر
با استفاده از یک توری پراش به عنوان عنصر اصلی پراکننده طول موج، تمام بخش های طیف، از مادون قرمز دور تا فرابنفش دور، قابل آنالیز هستند. توجه داشته باشید که افزایش تعداد خطوط موجود در یک توری پراش، دقت پراکندگی نور را افزایش می دهد و بر همین اساس، انواع مختلفی دارند.
توری پراش نوع مقعر، پراش و تصویر برداری را در یک عنصر ترکیب می کند و معمولاً در یک دستگاه اسپکتروفتومتر که از آشکارساز آرایه ای استفاده می کند، به کار می رود و از مزایای جالب توجه آن، در کنار استحکام، ابعاد کوچک و حذف قطعات متحرک، می توان به اندازه گیری موازی کل طیف اشاره کرد که باعث کاهش بسیار در زمان اندازه گیری می شود.
در صورت استفاده از توری پراش نوع صفحه ای یا تخت، لازم است از عناصر نوری اضافی (آینه های کروی یا عدسی) برای جهت دهی یا متمرکز نمودن پرتو استفاده شود.
پس از مرحله قبل، نور تفکیک شده بر روی یک آشکارساز یا طیف نگار ثبت می شود و برای هر طول موج متفاوت، نور عبوری از شکاف بر روی آشکارساز، در موقعیت و شدتی متفاوت تصویر می شود.
اگر برای این منظور، از یک آشکارساز منفرد مانند فتودیود استفاده شود، می توان طیف حاصل از توری پراش را به صورت مرحله ای اسکن کرد (معمولاً توری پراش می چرخد) که البته زمان اندازه گیری ممکن است زیاد باشد.
سیستم اسپکتروفتومتر دارای آشکارساز منفرد و توری چرخان
در اسپکتروفتومتر های مدرن معمولاً از آشکارساز های آرایه ای، مانند سنسور های بار جفت شده (CCD) و CMOS ها استفاده می شود که مشکل اسکن طولانی مدت را حذف می کنند. در چنین سیستم هایی، توری ثابت است و شدت هر طول موج نور توسط یک پیکسل متفاوت آرایه و به طور همزمان اندازه گیری می شود.
آرایه های CMOS و CCD حساسیت و سرعت بالاتری در آشکارسازی دارند، اما آرایه های دیودی دینامیک بسیار بهتری را ارائه می دهند. از این رو، انتخاب مناسب آشکارساز برای یک کاربرد خاص، می تواند عملکرد کل سیستم را بهبود بخشد.
سیستم کلی آشکارسازهای آرایه ای
به دلیل آرایه ای بودن این نوع آشکارساز ها، تمامی طول موج ها به طور همزمان اسکن می شوند و در نتیجه، هم زمان اندازه گیری و هم دقت اندازه گیری به طور قابل توجهی کاهش می یابد.
انواع مختلفی از لنز یا عدسی در دستگاه اسپکتروفتومتر استفاده می شود که هرکدام عملکرد منحصر به فرد خود را دارند. پیکربندی خاص و انواع لنزهای مورد استفاده بسته به طراحی و هدف اسپکتروفتومتری متفاوت است.
انواع عدسی در یک اسپکتروفتومتر
برخی از انواع لنزها یا عدسی های مورد استفاده در اسپکتروفتومترها عبارت اند از :
لنزهای کندانسور و استوانه ای
به طور کلی، معیارهای تعریف شده عملکرد ابزاری یک دستگاه اسپکتروفتومتر موارد زیر هستند:
مهم است که این پارامتر ها، به صورت دوره ای بررسی شوند. البته یک دستگاه اسپکتروفتومتر مدرن، اغلب دارای تست های خود تشخیصی عملکرد، تحت عنوان کالیبراسیون هستند.
یک دستگاه اسپکتروفتومتر مجهز به آشکارساز آرایه ای، بسته به نوع منبع نوری که در آن تعبیه می گردد، طیف وسیعی از طول موج ها، از فرابنفش تا نور مرئی و فروسرخ را شامل می شود.
منبع نور ایده آل، منبعی است که در تمامی طولموج های تحت پوشش خود، یک شدت ثابت داشته باشد و همچنین در طولانی مدت دارای پایداری و نویز کم باشد.
در یک دستگاه اسپکتروفتومتر UV-VIS-NIR، معمولاً از دو لامپ تنگستن و نوع دوتریومی استفاده می شود و در حالت های مختلفی مورد استفاده قرار می گیرند:
دقت داشته باشید که در طراحی یک سیستم اسپکترفتومتر، اندازه دستگاه متناسب با محدوده طول موجی و وضوح نوری (رزولوشن) تعیین می شود.
منابع نوری مختلف و محدوده طول موجی قابل ارائه
تعاریف متعددی برای پهنای باند یک دستگاه اسپکتروفتومتر استفاده می شود:
طبق تعریف دوم، پهنای باند طیفی یک اسپکتروفتومتر تابعی از طراحی نوری، توان تفکیک رزولوشن توری پراش و هندسه پرتو است. در ابزارهای با کیفیت بالاتر، می توان با تغییر شکاف بین شکاف های خروجی پهنای باند طیفی را تنظیم کرد.
برای دو خط طیفی متناظر با دو طول موج کنار هم ( λ و λ+Δλ )، تفکیک یا رزولوشن مقیاسی از گستردگی یا توزیع طیف است، به گونه ای که هنوز بتوان با کاهش آن، دو خط را تشخیص داد.
مطابق معیار توزیع رایلی، زمانی دو خط تفکیک پذیر نامیده می شوند که حداکثر کمینه خط اول ( λ) بر بیشینه خط دوم (λ+Δλ) منطبق شده باشد (شکل 8).
تفاوت تعریف رزولوشن بر اساس معیار رایلی و FWHM
در این شرایط، قدرت تفکیک توری با عرض W به صورت زیر تعریف می شود:
λ/Δλ=mN×W
در اینجا، N×W تعداد کل خطوط توری است که توسط نور پوشانده می شود. توجه داشته باشید که نمی توان m (مرتبه پراش) و N×W را مستقل از هم دانست. با این حال، علاوه بر کیفیت اپتیکی سطح توری و موقعیت شکاف، کیفیت اپتیک های به کار رفته در دستگاه اسپکتروفتومتر و اندازه های شکاف ورودی و خروجی نیز نقشی اساسی دارند.
طبق تعریف FWHM، رزولوشن یک دستگاه اسپکتروفتومتر بر حسب عرض شکاف ورودی (W)، فاصله کانونی سیستم نوری و پراکندگی خطی توری (dl/dλ) پراش تعیین می شود:
Δλ=W.dλ/dl
λ/Δλ=W.dl ⇒
این معادله زمانی استفاده شود که عرض شکاف ورودی برابر با عرض شکاف خروجی باشد. هرچه عرض شکاف کمتر باشد، رزولوشن بالاتر خواهد بود و خطا را کاهش می دهند. عرض شکاف متناسب با عرض باند طیفی است و هر تغییر در عرض شکاف را می توان به عنوان یک سطح از پیش تعیین شده پهنای باند طیفی تعیین کرد.
تأثیر رزولوشن بالا بر تفکیک نتایج طیفی
کاهش عرض شکاف، انرژی اپتیکی را در هنگام ورود به دستگاه اسپکتروفتومتر کاهش می دهد که می تواند میزان حساسیت دستگاه را مختل کند.
بیشترین تأثیر پهنای طیفی در هنگام اندازه گیری قله های بسیار باریک و تیز یا پیچیده یا جایی که دو یا چند قله بسیار نزدیک به هم هستند، مشهود می شود.
مقایسه پهنای باندهای مختلف
معمولاً این پارامتر با وضوح پیکسل اشتباه گرفته می شود. وضوح پیکسل یا دیجیتال، پهنای طیفی است که توسط یک پیکسل آرایه تشخیص داده می شود و با عرض پیکسل و پراکندگی طیف در آشکارسازهای آرایه ای تعیین می شود.
در یک دستگاه اسپکتروفتومتر، یک آشکارساز به کل انرژی نوری که به آن می رسد، بدون تبعیض بین طول موج های منفرد، پاسخ می دهد و نور سرگردان (Stray Light)، تابش طول موج های کاذب (به جز طول موج های انتخاب شده) است که به آشکارساز برخورد می کند.
مهم ترین منبع خطای مرتبط با ابزار، نور سرگردان است که بر دقت سیستم اندازه گیری طیف سنجی تأثیر می گذارد و معمولاً ناشی از نقص توری پراش، گرد و غبار، اجزای آسیب دیده یا فرسوده، بازتاب محفظه دستگاه اسپکتروفتومتر یا خطاهای سایر عناصر نوری است.
نور سرگردان
این اثر در غلظت های بالاتر، قابل توجه تر میشود زیرا در چنین غلظت هایی، نور سرگردان سهم بیشتری از کل نور دریافتی آشکارساز را تشکیل می دهد. به عبارت دیگر، در سطوح توان پایین، نور سرگردان باعث غیر خطی بودن سیگنال می شود و بنابراین محدوده اندازه گیری سیستم را محدود می کند.
میزان نور سرگردان، از روش های مختلفی تعیین می گردد:
چنین فیلترهایی، نور را در طول موجی که اندازه گیری باید انجام شود، جذب می کنند اما طول موج های بالاتری را ارسال می کنند. بنابراین هر گونه عبور نور به زیر طول موج قطع فیلترها، ناشی از نور سرگردان خواهد بود.
طبق تعریف، نور سرگردان نسبت عبور نور در ناحیه طول موج مسدود شده در زیر لبه فیلتر به میزان عبور نور در ناحیه غیر مسدود است.
شدت های طول موج لیزر (تک رنگ) و طول موج دیگر ( بااختلاف 10 نانومتر از طول موج لیزر) اندازه گیری می شود که نسبت دومی به اولی، معیاری برای نور سرگردان سیستم است.
تأثیر نور سرگردان بر نتایج طیفی
تکرارپذیری طول موج، به توانایی یک دستگاه اسپکتروفتومتر برای تولید نتایج ثابت، در حین اندازه گیری چندین باره یک طول موج (در مدت 1 دقیقه) اشاره دارد و یک ویژگی مهم برای ابزارهای مورد استفاده در طیف سنجی است.
در حقیقت، تکرارپذیری طول موج، ثبات اندازه گیری طول موج است و عوامل متعددی وجود دارد که می تواند بر تکرارپذیری طول موج تأثیر بگذارد، از جمله:
برای دستیابی به تکرار پذیری طول موج بالا، دستگاه اسپکتروفتومتر باید دارای اجزای پایدار و دقیقی باشد (شامل منابع نور، اجزای نوری با کیفیت بالا و آشکارسازها) که بتواند طول موج مورد نظر را در طول زمان حفظ کند. هر گونه نوسان یا ناپایداری در این اجزا می تواند باعث تغییر در طول موج اندازه گیری و عدم تکرارپذیری شود.
برای بررسی تکرار پذیری طول موج در یک دستگاه اسپکتروفتومتر، معمولاً موارد زیر انجام می گردد:
انحراف معیار کمتر، تکرارپذیری بهتر
بهبود تکرارپذیری طول موج را می توان از طریق روش های زیر به دست آورد:
کالیبراسیون های منظم را با استفاده از یک ماده مرجع شناخته شده انجام دهید تا مطمئن شوید که دستگاه اندازه گیری های دقیقی را ارائه می دهد. بررسی کالیبراسیون بر روی دستگاه، با استفاده از استاندارد های شناخته شده، مانند خطوط انتشار اتمی یا ویژگی های جذب در گازها، معمول است.
ابزار را در محیطی پایدار با دما، رطوبت و ارتعاش کنترل شده نگه دارید زیرا این کار، تغییرات ناشی از عوامل محیطی را به حداقل می رساند.
تعمیر، نگهداری منظم، تمیز کردن ابزار و تعویض اجزای فرسوده نیز می تواند در به حداقل رساندن هرگونه تغییر کمک کند.
اطمینان حاصل کنید که اپراتورها برای استفاده از ابزار و پیروی از روش های عملیاتی استاندارد، به درستی آموزش دیده اند.
بررسی های معمول کنترل کیفیت را انجام دهید تا مطمئن شوید که دستگاه مطابق با مشخصات کار می کند. داشتن یک فرآیند کنترل کیفیت قوی میتواند این اطمینان را حاصل کند که اندازهگیری ها در طول زمان سازگار و دقیق هستند.
دقت فتومتریک، به میزان دقت اندازه گیری روشنایی یا شدت نور اشاره دارد. به عبارت دیگر، دقت فوتومتریک بیان می کند که یک دستگاه اسپکتروفتومتر چقدر می تواند کل میزان نوری را که بر روی آشکارساز می تابد را با دقت اندازه گیری کند. به عبارت دیگر، دقت فوتومتریک تفاوت بین میزان جذب اندازه گیری شده و مقدار استاندارد تعیین شده است.
عواملی که می توانند بر دقت فتومتریک یک دستگاه اسپکتروفتومتر تأثیر بگذارند عبارت اند از:
دقت فتومتریک یک سیستم اسپکتروفتومتر را می توان با مقایسه داده های طیفی به دست آمده از دستگاه و مقادیر استاندارد شناخته شده اندازه گیری کرد که روش اندازه گیری، معمولاً شامل مراحل زیر است:
برای اندازه گیری دقت فوتومتریک، یک منبع نور کالیبره شده (مانند یک لامپ تنگستن هالوژن یا یک لامپ دوتریوم) به عنوان استاندارد مرجع مورد نیاز است.
با استفاده از منبع نور کالیبره شده به عنوان ورودی، طیف مرجع را با دستگاه اسپکتروفتومتر اندازه گیری کنید. طیف مرجع باید نشان دهنده خروجی واقعی منبع نور باشد.
طیف مرجع را با مقادیر استاندارد شناخته شده مقایسه کنید و فاکتورهای تصحیح مورد نیاز برای تنظیم خوانش های اندازه گیری طیفی را محاسبه کنید.
طیف نمونه را با استفاده از تنظیمات و شرایط طیف سنج مشابه اندازه گیری مرجع اندازه گیری کنید.
ضریب تصحیح محاسبه شده در مرحله 3 را برای اندازه گیری های به دست آمده در مرحله 4 اعمال کنید تا داده های فتومتریک تصحیح شده را به دست آورید.
نمودار مقایسه مقادیر دقت فتومتریک با مراجع استاندارد
میزان جذب (A)، که با عنوان چگالی نوری (OD) نیز شناخته می شود، به مقدار نور جذب شده توسط یک محلول نمونه گفته می شود. در یک دستگاه اسپکتروفتومتر، حد جذب یا محدودیت چگالی نوری به حداکثر مقدار نوری اطلاق می شود که می تواند توسط محلول نمونه جذب شود، پیش از اینکه آشکارساز اشباع شود و سیگنال دیگر خطی نباشد.
ارتباط مقادیر جذب و عبور در یک اسپکتروفتومتر
در اسپکتروفتومتری، اشباع آشکارساز زمانی اتفاق می افتد که شدت نور ورودی، از حداکثر قابلیت تشخیص آشکارساز بیشتر شود و زمانی اتفاق می افتد که سیگنال خیلی قوی باشد. در این صورت، آشکارساز اندازه گیری دقیقی نخواهد داشت و بنابراین می تواند منجر به مخدوش شدن یا از بین رفتن بخشی از داده ها شود.
تأثیر اشباع آشکارساز در اندازه گیری طیفی، قویاً به نوع آشکارساز مورد استفاده و نحوه عملکرد آن در ثبت سیگنال ها بستگی دارد. این فرایند اشباع می تواند به اثرات Blooming منجر شود، جایی که بار به پیکسل های مجاور سرازیر می شود و منجر به تخمین بیش از حد سیگنال می گردد.
در برخی از آشکارسازها، مانند سنسورهای با بار جفت شده (CCD)، اشباع می تواند باعث قطع شدن شدید سیگنال شود و در نتیجه اطلاعات مربوط به شدت نور فرودی از بین برود.
اشباع آشکارساز و اختلال در نتایج
برای جلوگیری از اشباع آشکارساز، مهم است که از آشکارسازهایی استفاده کنید که محدوده دینامیکی به اندازه کافی بالایی برای کنترل سطوح سیگنال مورد انتظار داشته باشند. علاوه بر این، تضعیف یا فیلتر مناسب نور ورودی نیز می تواند به کاهش شدت سیگنال دریافتی آشکارساز کمک کند.
اطمینان از رقیق شدن مناسب نمونه یا در صورت امکان استفاده از کووت هایی با طول مسیر کوتاه تر نیز مهم است. با رسیدن به مقدار حدی جذب، مقادیر جذب اندازه گیری شده به صورت غیرخطی تبدیل می شود و تعیین دقیق غلظت نمونه را غیرممکن می کند و دیگر نمی توان از قانون بیر-لامبرت استفاده کرد.
محدوده دینامیک خطی یک آشکارساز
بنابراین، مهم است که محدودیت های دینامیکی اسپکتروفتومتر را به دقت در نظر بگیرید و گام های مناسب برای بهینه سازی اندازه گیری هر نمونه جداگانه بردارید.
نسبت سیگنال به نویز (SNR) یک فاکتور بسیار مهم در اسپکتروفتومتری است. توانایی اسپکتروفتومتر برای اندازه گیری دقیق، به کیفیت سیگنال بدست آمده از آشکارساز و مدارهای الکتریکی بعدی بستگی دارد.
S/N نشان دهنده شدت سیگنال S تقسیم بر عرض نویز N خط مبنا است. به عبارت دیگر، مقادیر بزرگ تر S/N قدرت سیگنال بهتری را نسبت به نویز نشان می دهد و حساسیت بهتری ایجاد می کند.
در حقیقت، نسبت سیگنال به نویز (SNR) معیاری از کیفیت سیگنال را ارائه می دهد. به عبارت دقیق تر، SNR میانگین توان موجود در سیگنال را با میانگین توان موجود در نویز مقایسه می کند، که شامل هر سیگنالی از منابعی غیر از منبع سیگنال هدف می شود.
نسبت سیگنال به نویز
در یک دستگاه اسپکتروفتومتری، سیگنال مورد نظر شامل توان نوری در یک طول موج معین است که توسط توری پراش به آشکارساز هدایت می شود و SNR از منابع متعددی اعم از الکتریکی و نوری ناشی می شود.
SNR بر اساس موارد زیر محاسبه می شود:
SNR = Ī2sig / σ2n = (RṖopt)2 / σ2 n1 + σ2 n2 + σ2 n3 +…… +σ2 nN
که در آن قدرت یا توان نوری متوسط در سیگنال مورد نظر است و به واریانس منبع i ام جریان نویز در A2 اشاره دارد که میانگین توان را در یک سیگنال تصادفی بر اساس نظریه تبدیل فوریه توصیف می کند.
به منظور بهبود SNR در یک دستگاه طیف سنجی، در امکان سنجی های طراحی باید قدرت سیگنال اندازه گیری را افزایش داد و در عین حال منابع نویز را تا حد امکان به حداقل رساند.
1-نویز تاریک: که معمولاً به آن نویز حرارتی نیز گفته می شود، از تغییر تعداد الکترون های موجود برای هدایت جریان، حتی بدون برخورد نور به آشکارساز، ناشی می شود. انرژی حرارتی حاصل از گرمایش محیط، حامل های اضافی را تولید می کند که در جریان نویز سهیم می شوند.
2-نویز شات: که از تغییر در تعداد فوتون هایی که به آشکارساز برخورد می کنند، حاصل می شود، با این فرض که تمام فوتون ها به الکترون هایی تبدیل می شوند که به جریان کمک می کنند. بنابراین، بزرگی نویز شات، به تابش توان نوری روی آشکارساز بستگی دارد.
بزرگی نسبی نویز شات و نویز حرارتی به شدت به دمای محیط، مقدار در آشکارسازی و مقاومت مورد استفاده برای تبدیل جریان الکتریکی به ولتاژ برای پردازش بستگی دارد.
3-نویز الکترونیکی اضافی: که گاهی اوقات به عنوان نویز بازخوانی نیز شناخته می شود، از مداری که مستقیماً در پشت آشکارساز قرار دارد، ایجاد می شود که فیلتر اولیه و مقیاس بندی سیگنال را فراهم می کند. این نویز را می توان به عنوان جریان نویز اضافی یا با استفاده از یک اندازه گیری مدل سازی کرد.
5-نویز الگوی ثابت: از تغییر در پاسخ به نور فرودی در یک آرایه آشکارساز ناشی می شود. این تغییرات عمدتاً از تفاوت های بازده کوانتومی ناشی از تفاوت ها در ناحیه دیافراگم و ضخامت آشکارسازهایی که در طول ساخت رخ داده است، نشأت می گیرد. تنها اسپکتروفتومتر هایی که از یک آرایه آشکارساز خطی برای تمایز بین طول موج ها استفاده می کنند، از این منبع نویز رنج می برند.
6-نویز حاصل از نور سرگردان: نقص در ساختار توری پراش و مسیریابی تصادفی نور از آینه های “خاموش” روی DMD می تواند منجر به ورود نور از طریق مسیرهایی به آشکارساز شود که حاوی هیچ نوع اطلاعات مفیدی نیست.
بنابراین، نور سرگردان به عنوان یک روشنایی پس زمینه عمل می کند که باید پیش از اینکه خروجی یک آشکارساز با اطمینان به عنوان یک سیگنال حامل اطلاعات شناسایی شود، از آن فراتر رود.
چندین روش طراحی و اندازه گیری، بر اساس ماهیت منابع نویز، میتواند SNR را در یک دستگاه اسپکتروفتومتر بهبود ببخشد و منجر به اندازه گیری با کیفیت بالاتر و دقیق تر شود.
افزایش توان عملیاتی سیستم نوری، قدرت سیگنال موجود در آشکارساز را افزایش می دهد. به علاوه، استفاده از گریتینگ های هولوگرافیک، که دارای عیوب بسیار کمتری نسبت به گریتینگ های خط دار هستند، می تواند نور سرگردان تولید شده توسط سیستم نوری را کاهش دهد.
یک خنک کننده ترمو الکتریک (TEC) متصل به هر آشکارساز، دمای موثر آشکارساز را کاهش می دهد و بنابراین می تواند تاثیر آن بر SNR را کاهش دهد. استفاده از TEC به ویژه در مورد برخورد سیگنال نوری کم بر روی آشکارساز مؤثر است، زیرا سیگنال آشکارساز در مدت زمان طولانی یکپارچه می شود تا کل قدرت سیگنال نوری جمع آوری شده را افزایش دهد.
از آنجایی که این فرآیند جریان های نویز را نیز یکپارچه می کند، قدرت نویز یکپارچه می تواند قدرت سیگنال بهبود یافته را تحت تأثیر قرار دهد. سیستم های TEC نویز را به حد کافی پایین می آورند و زمان های ادغام طولانی تر، در افزایش SNR موثر است.
در این مقاله، ابتدا تعریف دقیقی از دستگاه اسپکتروفتومتر ارائه دادیم و مکانیزم آنها را بر اساس دو دسته بندی نوع جذبی-عبوری و بازتابی توضیح دادیم.
سپس، عملکرد کلی دستگاه های اسپکتروفتومتر و اجزای تشکیل دهنده آن را شرح دادیم. در نهایت نیز پارامترهای کلیدی که در انتخاب یک دستگاه اسپکتروفتومتری موثر هستند را معرفی نمودیم.
اسپکتروفتومتر یک ابزار علمی است که برای اندازه گیری مقدار نور جذب شده یا ارسال شده توسط یک نمونه در طول موج های مختلف استفاده می شود. این به طور گسترده در زمینه های مختلف از جمله شیمی، بیوشیمی، علوم محیطی و پزشکی استفاده می شود.
امیدواریم که با خوندن این مقاله حوصله تون سر نرفته باشه ☺️
به شما کاربر عزیز پیشنهاد می کنیم که در صورت تمایل، محصولات شرکت دانش بنیان بلورآزما را نیز مشاهده کنید و در صورت نیاز، می توانید کاتالوگ هر محصول ( مثل ریجنت و… ) را به صورت جداگانه دانلود کنید🌷
Basics of spectral measurments, Issued by: JETI Technische Instrumente GmbH, Jena, May 2005
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